Marinopyrrole derivatives and methods of making and using same

ABSTRACT

Marinopyrrole derivatives and methods for their synthesis and use are described herein. Novel cyclic and symmetric marinopyrroles with triazole substituents having antibacterial activity against resistant bacterial strains, such as MRSA are introduced. Also provided are methods of using the compounds for treating or preventing cancer and/or microbial infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/941,374, filed Feb. 18, 2014, and U.S. Provisional Application No.61/981,357, filed Apr. 18, 2014, which are hereby incorporated herein byreference in their entireties.

FIELD

The subject matter disclosed herein generally relates to marinopyrrolederivatives and methods of use thereof.

BACKGROUND

Apoptosis is the best-characterized mode of physiological cell death,which plays an essential role in the development and homeostasis ofmulticellular organisms. Apoptosis is executed by caspases, a family ofcysteine proteases, whose activation is initiated via two majorpathways: the death receptor (extrinsic) pathway and the mitochondrial(intrinsic) pathway. The activated caspases cleave a number of cellularproteins to generate many of the hallmark morphological features ofapoptosis, including DNA fragmentation and membrane blebbing.

The Bcl-2 family of proteins plays a pivotal role in apoptosis byregulating the mitochondrial outer membrane permeabilization (MOMP).MOMP results in the release of apoptogenic factors (e.g., cytochrome cand Smac) from the mitochondria into the cytosol where they directlypromote caspase activation and subsequent cell death. Members of theBcl-2 family contain up to four evolutionarily conserved domains calledBcl-2 homology (BH) domains 1 to 4 and can be classified into threegroups based on their domain architecture and function in apoptosis:multidomain (BH1-4) anti-apoptotic Bcl-2 proteins (e.g., Bcl-2,Bcl-X_(L) and Mcl-1), multidomain (BH1-3) pro-apoptotic Bcl-2 proteins(e.g., Bax and Bak), and BH3-only Bcl-2 proteins (e.g., Bad, Bid, Bim,Noxa and Puma). Many of the Bcl-2 family proteins can interact with eachother to determine the cell fate. The three-dimensional structuresreveal that the BH1-3 domains of anti-apoptotic Bcl-2 proteins form ahydrophobic surface groove to which the BH3 domains of pro-apoptoticBcl-2 family members bind (Sattler et al., (1997) Science 275:983-986;Day et al., (2008) J Mol Biol 380:958-971). The multidomainpro-apoptotic Bcl-2 proteins Bax and Bak are two major effectors ofMOMP, which homo-oligomerize and form pores in the mitochondrial outermembrane to induce MOMP upon apoptotic stimulation. The anti-apoptoticBcl-2 proteins prevent MOMP by directly binding to both classes ofpro-apoptotic Bcl-2 proteins. In contrast, the BH3-only proteins triggerBax and Bak to induce MOMP. Based on their ability to interact with themultidomain anti- and pro-apoptotic Bcl-2 proteins, the BH3-onlyproteins are often further divided into two subgroups: direct activatorsand sensitizers/de-repressors. The direct activators, including Bid, Bimand Puma, are able to not only interact with and inhibit all theanti-apoptotic Bcl-2 proteins but also directly bind to and activate theeffectors Bax and Bak. On the other hand, the sensitizers/de-repressorsappear to function essentially as transdominant inhibitors by occupyingthe hydrophobic groove of anti-apoptotic Bcl-2 proteins, therebydisplacing the direct activators to promote MOMP and preventing anyfuture bindings of the direct activators or effectors to anti-apoptoticBcl-2 proteins. Moreover, unlike the direct activators, thesensitizers/de-repressors are more selective in binding to theanti-apoptotic Bcl-2 members. For example, Bad binds and antagonizesBcl-2 and Bcl-X_(L) but not Mcl-1, whereas Noxa binds and antagonizesMcl-1 but not Bcl-2 and Bcl-X_(L). This observation indicates that theBH3-only proteins provide a fine control of MOMP in a Bax/Bak-dependentmanner and opportunities to design specific inhibitors for each of theanti-apoptotic Bcl-2 family members.

The evasion of apoptosis is considered to be a hallmark of cancers and acause of resistance to radiation and chemotherapies. Consistently, highlevels of the anti-apoptotic Bcl-2 family proteins are associated withthe pathogenesis of cancer and resistance to therapy (Reed et al.,(1996) J Cell Biochem 60:23-32; Reed, (1997) Adv Pharmacol 41:501-532).A recent analysis of somatic copy-number alterations (SCNAs) showed thattwo anti-apoptotic Bcl-2 family genes (Bcl-X_(L) and Mcl-1) undergofrequent somatic amplifications in multiple cancers and that cancercells carrying Bcl-X_(L) and Mcl-1 amplifications are dependent on theexpression of these genes for survival (Beroukhim et al., (2010) Nature463:899-905). Thus, Bcl-X_(L) and Mcl-1 are very attractive targets forthe development of anticancer agents.

Over the last few years, several small-molecule Bcl-2 inhibitors havebeen synthesized as BH3 mimetics and some of these molecules haveentered clinical trials (Yip et al., (2008) Oncogene 27:6398-6406;Vogler et al., (2009) Cell Death Differ 16:360-367; Kazi et al., (2011)J Biol Chem 286:9382-9392). Although Bcl-2 and Bcl-X_(L) have been theprimary focus for the design of small-molecule inhibitors, recentstudies have demonstrated that Mcl-1 also plays an important role forcancer cell survival and that it is necessary to neutralize both arms ofthe anti-apoptotic Bcl-2 family (Bcl-2/Bcl-X_(L) and Mcl-1) forapoptosis to occur in many cell types (Willis et al., (2005) Genes Dev19:1294-1305).

To date, the most potent and selective small-molecule Bcl-2 inhibitorsare ABT-737 and its orally active analog ABT-263, which inhibit Bcl-2and Bcl-X_(L) at subnanomolar concentrations but only weakly targetMcl-1 (Tse et al., (2008) Cancer Res 68:3421-3428). Consequently, theseagents generally lack efficacy in cancers with elevated Mcl-1 and inmany instances this resistance can be overcome by downregulation ofMcl-1 (Id.; Oltersdorf et al., (2005) Nature 435:677-681; van Delft etal., (2006) Cancer Cell 10:389-399; Chen et al., (2007) Cancer Res 67,782-791; Konopleva et al., (2006) Cancer Cell 10:375-388; Lin et al.,(2007) Oncogene 26:3972-3979; Tahir et al., (2007) Cancer Res67:1176-1183). Moreover, it has recently been shown that cancer cellscan quickly acquire resistance to ABT-737 by upregulation of Mcl-1(Yecies et al., (2009) Blood 233-304; Hikita et al., (2010) Hepatology52:1310-1321). What are thus needed are compounds that specifically bindto Mcl-1 to overcome such resistance. Such compounds can be used totreat and/or prevent various cancers. The compositions and methodsdisclosed herein address these and other needs.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, kits, and methods, as embodied and broadly describedherein, the disclosed subject matter relates to compositions, methods ofmaking said compositions, and methods of using said compositions. Morespecifically, marinopyrrole derivatives are provided herein. Alsodisclosed herein are methods of use of the marinopyrrole derivatives asanticancer agents. Also disclosed herein are methods of use of themarinopyrrole derivatives as antimicrobial agents. Also disclosed hereinare methods of use of the marinopyrrole derivatives as antibacterialagents.

Methods of making and using marinopyrrole derivatives are also disclosedherein.

Additional advantages will be set forth in part in the description thatfollows or may be learned by practice of the aspects described below.The advantages described below will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 shows the structure of marinopyrrole A (1) and cyclicmarinopyrroles 3-8.

FIG. 2 shows the ELISA and physicochemical properties of 1 andsymmetrical marinopyrroles.

FIG. 3 shows the ELISA and physicochemical properties of cyclicmarinopyrroles

FIG. 4 shows the effect of marinopyrrole derivatives on Mcl-1, Bim andcaspase 3 in human breast cancer cells.

FIG. 5 displays that compounds II-4 and I-20 directly bind to Mcl-1 asmeasured by fluorescence quenching.

FIG. 6 (A) displays the overlaid ¹H-¹⁵N HSQC spectra of Mcl-1,¹⁵N-Mcl-1:II-4 and of ¹⁵N-Mcl-1:I-20 at 1:1 stoichiometric ratio. FIG. 6(B) displays a structural model of Mcl-1 with a view of the BH3 pockethighlighting the position of residues undergoing significant chemicalshift perturbations. The NMR data are consistent with the II-4 and I-20binding to the BH3 pocket of Mcl-1. BIM BH3 bound to Mcl-1 is shown forcomparison. FIG. 6(C) displays cartoons highlighting the structure ofeach compound and specific interactions with the Mcl-1 binding site.

FIG. 7 displays Mcl-1/Bim and Bcl-xL/Bim ELISA assays (A). (B) showsresults from coimmunoprecipitation and densiometry quantitation of ofBim/Mcl-1 and Mim/Bcl-xL in MDA-MB-468 cells treated with 1 and I-2 andthe lysates.

FIG. 8 displays the western blots after MDA-MB-468 cells were treatedwith 1 and I-2 and processed.

FIG. 9 displays the MTT and soft agar (anchorage-independent growth)results for MDA-MB-468 and A-549 cells treated with 1 or I-2 in (A)96-well plate for 48 hours (MTT) and (B) 12-well plates for 21 days(soft agar).

FIG. 10 displays the average % change in (A) tumor size and (B) mouseweight in Nude mice bearing s/c/A-549 xenografts injected with vehicleor I-2 (4 mpk/day) for 14 days.

FIG. 11 displays standard curves for (A) (±)-1 and (B) I-2 as increasingamounts of (±)-1 and I-2 were mixed with mouse blood, extracted withmethanol, and injected into LC-MS using Agilent 1220 Infinity LC/6120Quadrupole LC/MS with Agilent Zorbax, SBC18 column.

FIG. 12 displays the % area absorbance as (±)-1 and I-2 were incubatedin 1 M NaOH for 1, 2, 4, 8, 24, and 48 hours at room temperature.

DETAILED DESCRIPTION

The materials, compounds, compositions, articles, and methods describedherein may be understood more readily by reference to the followingdetailed description of specific aspects of the disclosed subject matterand the Examples included therein.

Before the present materials, compounds, compositions, kits, and methodsare disclosed and described, it is to be understood that the aspectsdescribed below are not limited to specific synthetic methods orspecific reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

General Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions, reference to “thecompound” includes mixtures of two or more such compounds, reference to“an agent” includes mixture of two or more such agents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed, then“less than or equal to” the value, “greater than or equal to the value,”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed, then “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe application data are provided in a number of different formats andthat this data represent endpoints and starting points and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point “15” are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15. It is also understood that each unit betweentwo particular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals (e.g., cats, dogs, etc.),livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.“Subject” can also include a mammal, such as a primate or a human.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,tumor growth). It is understood that this is typically in relation tosome standard or expected value, in other words it is relative, but thatit is not always necessary for the standard or relative value to bereferred to. For example, “reduces tumor growth” means reducing the rateof growth of a tumor relative to a standard or a control. For example,“reduces bacterial infection” means reducing the spread of a bacterialinfection relative to a standard or a control.

By “prevent” or other forms of the word, such as “preventing” or“prevention,” is meant to stop a particular event or characteristic, tostabilize or delay the development or progression of a particular eventor characteristic, or to minimize the chances that a particular event orcharacteristic will occur. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce. Asused herein, something could be reduced but not prevented, but somethingthat is reduced could also be prevented. Likewise, something could beprevented but not reduced, but something that is prevented could also bereduced. It is understood that where reduce or prevent are used, unlessspecifically indicated otherwise, the use of the other word is alsoexpressly disclosed.

By “treat” or other forms of the word, such as “treated” or “treatment,”is meant to administer a composition or to perform a method in order toreduce, prevent, inhibit, or eliminate a particular characteristic orevent (e.g., tumor growth or survival). The term “control” is usedsynonymously with the term “treat.”

The term “anticancer” refers to the ability to treat or control cellularproliferation and/or tumor growth at any concentration.

By “antimicrobial” is meant the ability to treat or control (e.g.,reduce, prevent, or eliminate) the growth of a microbe at anyconcentration. Similarly, the term “antibacterial” refers to the abilityto treat or control cellular bacteria growth at any concentration.

It is understood that throughout this specification the identifiers“first” and “second” are used solely to aid in distinguishing thevarious components and steps of the disclosed subject matter. Theidentifiers “first” and “second” are not intended to imply anyparticular order, amount, preference, or importance to the components orsteps modified by these terms.

Chemical Definitions

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

“Z¹,” “Z²,” “Z³,” and “Z⁴” are used herein as generic symbols torepresent various specific substituents. These symbols can be anysubstituent, not limited to those disclosed herein, and when they aredefined to be certain substituents in one instance, they can, in anotherinstance, be defined as some other substituents.

The term “aliphatic” as used herein refers to a non-aromatic hydrocarbongroup and includes branched and unbranched, alkyl, alkenyl, or alkynylgroups.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl,tetracosyl, and the like. The alkyl group can also be substituted orunsubstituted. The alkyl group can be substituted with one or moregroups including, but not limited to, alkyl, halogenated alkyl, alkoxy,alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid,ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo,sulfonyl, sulfone, sulfoxide, or thiol, as described below.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “alkoxy” as used herein is an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group can bedefined as —OZ¹ where Z¹ is alkyl as defined above.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (Z¹Z²)C═C(Z³Z⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol, as described below.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be substituted with oneor more groups including, but not limited to, alkyl, halogenated alkyl,alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylicacid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo,sulfonyl, sulfone, sulfoxide, or thiol, as described below.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “heteroaryl” isdefined as a group that contains an aromatic group that has at least oneheteroatom incorporated within the ring of the aromatic group. Examplesof heteroatoms include, but are not limited to, nitrogen, oxygen,sulfur, and phosphorus. The term “non-heteroaryl,” which is included inthe term “aryl,” defines a group that contains an aromatic group thatdoes not contain a heteroatom. The aryl or heteroaryl group can besubstituted or unsubstituted. The aryl or heteroaryl group can besubstituted with one or more groups including, but not limited to,alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol asdescribed herein. The term “biaryl” is a specific type of aryl group andis included in the definition of aryl. Biaryl refers to two aryl groupsthat are bound together via a fused ring structure, as in naphthalene,or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group asdefined above where at least one of the carbon atoms of the ring issubstituted with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkylgroup can be substituted or unsubstituted. The cycloalkyl group andheterocycloalkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onedouble bound, i.e., C═C. Examples of cycloalkenyl groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term“heterocycloalkenyl” is a type of cycloalkenyl group as defined above,and is included within the meaning of the term “cycloalkenyl,” where atleast one of the carbon atoms of the ring is substituted with aheteroatom such as, but not limited to, nitrogen, oxygen, sulfur, orphosphorus. The cycloalkenyl group and heterocycloalkenyl group can besubstituted or unsubstituted. The cycloalkenyl group andheterocycloalkenyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol as described herein.

The term “cyclic group” is used herein to refer to either aryl groups,non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl groups), or both. Cyclic groups have one or more ringsystems that can be substituted or unsubstituted. A cyclic group cancontain one or more aryl groups, one or more non-aryl groups, or one ormore aryl groups and one or more non-aryl groups.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” or “CO” is a short hand notationfor C═O.

The terms “amine” or “amino” as used herein are represented by theformula —NZ¹Z², where Z¹ and Z² can each be substitution group asdescribed herein, such as hydrogen, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH. A “carboxylate” or “carboxyl” group as used herein isrepresented by the formula —C(O)O⁻.

The term “ester” as used herein is represented by the formula —OC(O)Z¹or —C(O)OZ¹, where Z¹ can be an alkyl, halogenated alkyl, alkenyl,alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl,or heterocycloalkenyl group described above.

The term “ether” as used herein is represented by the formula Z¹OZ²,where Z¹ and Z² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ketone” as used herein is represented by the formula Z¹C(O)Z²,where Z¹ and Z² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “halide” or “halogen” as used herein refers to the fluorine,chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “silyl” as used herein is represented by the formula —SiZ¹Z²Z³,where Z¹, Z², and Z³ can be, independently, hydrogen, alkyl, halogenatedalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group describedabove.

The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula —S(O)₂Z¹, where Z¹ can be hydrogen, an alkyl,halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group describedabove.

The term “sulfonylamino” or “sulfonamide” as used herein is representedby the formula —S(O)₂NH—.

The term “phosphonyl” is used herein to refer to the phospho-oxo grouprepresented by the formula —P(O)(OZ¹)₂, where Z¹ can be hydrogen, analkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl,cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groupdescribed above.

The term “thiol” as used herein is represented by the formula —SH.

The term “thio” as used herein is represented by the formula —S—.

“R¹,” “R²,” “R³,” “R^(n),” etc., where n is some integer, as used hereincan, independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an amine group, an alkyl group, a halide, andthe like. Depending upon the groups that are selected, a first group canbe incorporated within second group or, alternatively, the first groupcan be pendant (i.e., attached) to the second group. For example, withthe phrase “an alkyl group comprising an amino group,” the amino groupcan be incorporated within the backbone of the alkyl group.Alternatively, the amino group can be attached to the backbone of thealkyl group. The nature of the group(s) that is (are) selected willdetermine if the first group is embedded or attached to the secondgroup.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

The following abbreviations are used herein: ADME, absorption,distribution, metabolism and excretion; DCM, dichloromethane; DPPP,bis(diphenylphosphino)propane; DIEA, diisopropylethylamine; DMF,dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; ESI,electrospray ionization; IBX, 2-iodoxybenzoic acid; KBr, potassiumbromide; KF, potassium fluoride; MeCN, acetonitrile; MeOH, methylalcohol; NCS, N-chlorosuccinimide; SAR, Structure Activity Relationship;TBAF, tetrabutylammonium fluoride; TBDMS, t-butyldimethylsilyl; TBDMSCl,t-butyldimethylsilyl chloride; Tf, trifluoromethanesulfonyl; THF,tetrahydrofuran.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples andFigures.

Compounds

Marinopyrrole derivatives are described herein. The marinopyrrolederivatives can have the following Formula I:

-   wherein R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, and R¹⁴ are    independently chosen from hydrogen, halogen, hydroxyl, cyano, nitro,    sulfonyl, phosphonyl, substituted or unsubstituted amino,    substituted or unsubstituted alkyl, substituted or unsubstituted    cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or    unsubstituted heterocycloalkyl, substituted or unsubstituted    alkenyl, substituted or unsubstituted cycloalkenyl, substituted or    unsubstituted heteroalkenyl, substituted or unsubstituted    heterocycloalkenyl, substituted or unsubstituted alkynyl,    substituted or unsubstituted heteroalkynyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted alkoxyl, substituted or unsubstituted    aryloxyl, and substituted or unsubstituted carboxyl;-   R⁵ and R¹² are independently chosen from hydrogen, halogen,    substituted or unsubstituted alkyl, substituted or unsubstituted    heteroalkyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, and a monovalent cation-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula I, adjacent R groups, e.g., R¹ and R², canbe combined to form a substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkenyl, substituted or unsubstitutedcycloalkynyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted heterocycloalkenyl, or substituted or unsubstitutedheterocycloalkynyl. For example, R¹ can be a substituted orunsubstituted ethylene group and R² can be a substituted orunsubstituted propylene group that combine to form a substituted orunsubstituted phenyl. Other adjacent R groups include the combinationsof R² and R³; R³ and R⁴; R⁶ and R⁷; R⁸ and R⁹; R⁹ and R¹⁰; R¹⁰ and R¹¹;and R¹⁴ and R¹⁵.

In some examples of Formula I, the marinopyrrole derivatives can be ofFormula II:

-   wherein R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are    each independently selected from hydrogen, halogen, hydroxyl, cyano,    nitro, sulfonyl, phosphonyl, carboxylate, substituted or    unsubstituted amino, substituted or unsubstituted alkyl, substituted    or unsubstituted heteroalkyl, substituted or unsubstituted alkenyl,    substituted or unsubstituted heteroalkenyl, substituted or    unsubstituted alkynyl, substituted or unsubstituted heteroalkynyl,    substituted or unsubstituted aryl, substituted or unsubstituted    heteroaryl, substituted or unsubstituted alkoxyl, substituted or    unsubstituted aryloxyl, and substituted or unsubstituted carboxyl;-   R⁵ is selected from hydrogen, substituted or unsubstituted alkyl,    substituted or unsubstituted heteroalkyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl, and a    monovalent cation;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

Examples of monovalent cations include, but are not limited to Na⁺, Li⁺,K⁺, and NH₄ ⁺.

In some examples of Formula II, R¹, R², R³, R⁴, R⁵, R⁹, R¹⁰, and R¹¹ areindependently selected from hydrogen, halogen, hydroxyl, sulfonyl,phosphonyl, carboxylate, substituted or unsubstituted amino, andsubstituted or unsubstituted carboxyl. In some examples of Formula II,R¹, R², R³, R⁴, R⁸, R⁹, R¹⁰, and R¹¹ are independently selected fromhydrogen, OCH₃, OR¹⁶, NH₂, NHR¹⁶, S(O)₂R¹⁶, P(O)(OR¹⁶)₂, and CO₂R¹⁶,wherein R¹⁶ is H, substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

In some examples of Formula II, one or more of R¹, R², R³, R⁴, R⁸, R⁹,R¹⁰, and R¹¹ is a halogen (e.g., F, Cl, Br, I). In some examples ofFormula II, one of R¹, R², R³, and R⁴ is a halogen (e.g., F, Cl, Br, I).

In some examples of Formula II, R⁶, R⁷, R¹⁴ and R¹⁵ are independentlyselected from hydrogen, halogen (e.g., F, Cl, Br, I) and hydroxyl. Insome examples of Formula II, one of R⁶, R⁷, R¹⁴ and R¹⁵ is a halogen(e.g., F, Cl, Br, I). In some examples of Formula II, R⁶, R⁷, R¹⁴ andR¹⁵ are independently selected from F, Cl, Br, and I.

In some examples of Formula II, R⁵ is selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteralkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, and a monovalent cation (e.g., Na⁺, Li⁺, K⁺,or NH₄ ⁺).

In some examples of Formula II, adjacent R groups, e.g., R¹ and R², canbe combined to form a substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkenyl, substituted or unsubstitutedcycloalkynyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted heterocycloalkenyl, or substituted or unsubstitutedheterocycloalkynyl. For example, R¹ can be a substituted orunsubstituted ethylene group and R² can be a substituted orunsubstituted propylene group that combine to form a substituted orunsubstituted phenyl. Other adjacent R groups include the combinationsof R² and R³; R³ and R⁴; R⁶ and R⁷; R⁸ and R⁹; R⁹ and R¹⁰; R¹⁰ and R¹¹;and R¹⁴ and R¹⁵.

In some examples of Formula II, R¹⁵ is hydrogen.

In some examples of Formula II, the compounds are of Formula II-1:

-   wherein R¹, R², R³, R⁴, R⁸, R⁹, R¹⁰, and R¹¹ are independently    selected from hydrogen, halogen, hydroxyl, cyano, nitro, sulfonyl,    phosphonyl, substituted or unsubstituted amino, substituted or    unsubstituted alkyl, substituted or unsubstituted heteroalkyl,    substituted or unsubstituted alkenyl, substituted or unsubstituted    heteroalkenyl, substituted or unsubstituted alkynyl, substituted or    unsubstituted heteroalkynyl, substituted or unsubstituted aryl,    substituted or unsubstituted heteroaryl, substituted or    unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, and    substituted or unsubstituted carboxyl;-   R⁶, R⁷, and R¹⁴ are independently selected from F, Cl, Br, and I;-   R⁵ is selected from hydrogen, substituted or unsubstituted alkyl,    substituted or unsubstituted heteroalkyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl, and a    monovalent cation;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula II, one or more of R¹, R², R⁴, R⁸, R⁹, andR¹¹ is hydrogen.

In some examples of Formula II, the compounds are of Formula III:

-   wherein R³, R⁶, R⁷, R¹⁰, R¹⁴, and R¹⁵ are independently selected    from hydrogen, halogen, hydroxyl, cyano, nitro, sulfonyl,    phosphonyl, substituted or unsubstituted amino, substituted or    unsubstituted alkyl, substituted or unsubstituted heteroalkyl,    substituted or unsubstituted alkenyl, substituted or unsubstituted    heteroalkenyl, substituted or unsubstituted alkynyl, substituted or    unsubstituted heteroalkynyl, substituted or unsubstituted aryl,    substituted or unsubstituted heteroaryl, substituted or    unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, and    substituted or unsubstituted carboxyl;-   R⁵ is selected from hydrogen, substituted or unsubstituted alkyl,    substituted or unsubstituted heteroalkyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl, and a    monovalent cation;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula III, R³ and R¹⁰ are independently selectedfrom hydrogen, halogen, hydroxyl, sulfonyl, phosphonyl, carboxylate,substituted or unsubstituted amino, and substituted or unsubstitutedcarboxyl. In some examples of Formula III, R³ and R¹⁰ are independentlyselected from hydrogen, OCH₃, OR¹⁶, NH₂, NHR¹⁶, S(O)₂R¹⁶, P(O)(OR¹⁶)₂,and CO₂R¹⁶, wherein R¹⁶ is H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedherteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

In some examples of Formula III, the compounds are of Formula III-1:

-   wherein R³, and R¹⁰ are independently selected from hydrogen,    halogen, hydroxyl, cyano, nitro, sulfonyl, phosphonyl, substituted    or unsubstituted amino, substituted or unsubstituted alkyl,    substituted or unsubstituted heteroalkyl, substituted or    unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl,    substituted or unsubstituted alkynyl, substituted or unsubstituted    heteroalkynyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted alkoxyl,    substituted or unsubstituted aryloxyl, and substituted or    unsubstituted carboxyl;-   R⁶, R⁷, and R¹⁴, are independently selected from F, Cl, Br, and I;-   R⁵ is selected from hydrogen, substituted or unsubstituted alkyl,    substituted or unsubstituted heteroalkyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl, and a    monovalent cation;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula III, the compounds are of Formula III-2:

wherein R³, R⁶, R⁷, R¹⁰, R¹⁴ and R¹⁵ are independently selected fromhydrogen, halogen, hydroxyl, cyano, nitro, sulfonyl, phosphonyl,substituted or unsubstituted amino, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted heteroalkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted heteroalkynyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted alkoxyl, substituted orunsubstituted aryloxyl, and substituted or unsubstituted carboxyl;and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula III, the compounds are of Formula III-3:

-   wherein R³ and R¹⁰ are independently selected from halogen,    hydroxyl, cyano, nitro, sulfonyl, phosphonyl, substituted or    unsubstituted amino, substituted or unsubstituted alkyl, substituted    or unsubstituted heteroalkyl, substituted or unsubstituted alkenyl,    substituted or unsubstituted heteroalkenyl, substituted or    unsubstituted alkynyl, substituted or unsubstituted heteroalkynyl,    substituted or unsubstituted aryl, substituted or unsubstituted    heteroaryl, substituted or unsubstituted alkoxyl, substituted or    unsubstituted aryloxyl, and substituted or unsubstituted carboxyl;-   R⁶, R⁷, and R¹⁴, are independently selected from F, Cl, Br, and I;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula III-3, one or more of R⁶, R⁷, and R¹⁴ is Cl.In some examples of Formula III-3, R⁶ is Cl. In some examples of FormulaIII-3, R⁷ is Cl. In some examples of Formula III-3, R¹⁴ is Cl. In someexamples of Formula III-3, R⁶ and R⁷ are Cl. In some examples of FormulaIII-3, R⁷, and R¹⁴ are Cl. In some examples of Formula III-3, R⁶ and R¹⁴are Cl.

In some examples of Formula III-3, the compounds are of Formula III-4:

-   wherein R³ and R¹⁰ are independently selected from halogen,    hydroxyl, cyano, nitro, sulfonyl, phosphonyl, substituted or    unsubstituted amino, substituted or unsubstituted alkyl, substituted    or unsubstituted heteroalkyl, substituted or unsubstituted alkenyl,    substituted or unsubstituted heteroalkenyl, substituted or    unsubstituted alkynyl, substituted or unsubstituted heteroalkynyl,    substituted or unsubstituted aryl, substituted or unsubstituted    heteroaryl, substituted or unsubstituted alkoxyl, substituted or    unsubstituted aryloxyl, and substituted or unsubstituted carboxyl;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula III-4, the compounds are of Formula IV:

-   wherein R¹⁷ is selected from halogen, hydroxyl, cyano, nitro,    sulfonyl, phosphonyl, substituted or unsubstituted amino,    substituted or unsubstituted alkyl, substituted or unsubstituted    heteroalkyl, substituted or unsubstituted alkenyl, substituted or    unsubstituted heteroalkenyl, substituted or unsubstituted alkynyl,    substituted or unsubstituted heteroalkynyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted alkoxyl, substituted or unsubstituted    aryloxyl, and substituted or unsubstituted carboxyl;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula IV, R¹⁷ is selected from hydroxyl, sulfonyl,phosphonyl, and substituted or unsubstituted carboxyl. In some examplesof Formula IV, R¹⁷ is selected from hydroxyl, S(O)₂R¹⁶, P(O)(OR¹⁶)₂, andCO₂R¹⁶, wherein R¹⁶ is H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedherteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Insome examples of Formula IV, R¹⁷ is selected from SO₂CF₃, OH, CO₂CH₃,CO₂H, PO(OCH₂CH₃)₂, and PO(OH)₂.

In some examples of Formula IV, the compounds are of Formula IV-1:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula IV, the compounds are of Formula IV-2:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula IV, the compounds are of Formula IV-3:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula IV, the compounds are of Formula IV-4:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula IV, the compounds are of Formula IV-5:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula IV, the compounds are of Formula IV-6:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula I, the marinopyrrole derivatives can havethe following Formula V:

-   wherein R⁶, R⁷, R¹³, and R¹⁴ are independently chosen from hydrogen,    halogen, and hydroxyl;-   R⁵ and R¹² are independently chosen from hydrogen, halogen, and    substituted or unsubstituted alkyl;-   R², R⁹, R¹⁸ and R¹⁹ are independently chosen from hydrogen, halogen,    hydroxyl, cyano, nitro, sulfonyl, phosphonyl, substituted or    unsubstituted amino, substituted or unsubstituted alkyl, substituted    or unsubstituted cycloalkyl, substituted or unsubstituted    heteroalkyl, substituted or unsubstituted heterocycloalkyl,    substituted or unsubstituted alkenyl, substituted or unsubstituted    cycloalkenyl, substituted or unsubstituted heteroalkenyl,    substituted or unsubstituted heterocycloalkenyl, substituted or    unsubstituted alkynyl, substituted or unsubstituted heteroalkynyl,    substituted or unsubstituted aryl, substituted or unsubstituted    heteroaryl, substituted or unsubstituted alkoxyl, substituted or    unsubstituted aryloxyl, and substituted or unsubstituted carboxyl;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula V, R⁶, R⁷, R¹³ and R¹⁴ are eachindependently a halogen (e.g., F, Cl, Br, or I). In some examples ofFormula V, R⁶, R⁷, R¹³ and R¹⁴ are each Cl.

In some examples of Formula V, R⁵ and R¹² are each H.

In some examples of Formula V, where R⁶, R⁷, R¹³ and R¹⁴ are each Cl, R⁵and R¹² are each H, R² and R⁹ are each R^(a), and R¹⁸ and R¹⁹ are eachR^(b), the compounds are of Formula B:

-   wherein R^(a) and R^(b) are independently chosen from hydrogen,    halogen, hydroxyl, cyano, nitro, sulfonyl, phosphonyl, substituted    or unsubstituted amino, substituted or unsubstituted alkyl,    substituted or unsubstituted cycloalkyl, substituted or    unsubstituted heteroalkyl, substituted or unsubstituted    heterocycloalkyl, substituted or unsubstituted alkenyl, substituted    or unsubstituted cycloalkenyl, substituted or unsubstituted    heteroalkenyl, substituted or unsubstituted heterocycloalkenyl,    substituted or unsubstituted alkynyl, substituted or unsubstituted    heteroalkynyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted alkoxyl,    substituted or unsubstituted aryloxyl, and substituted or    unsubstituted carboxyl;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In some examples of Formula B, R^(a) is chosen from hydrogen and halogen(e.g., F, Cl, Br and I). In some examples of Formula B, R^(a) is chosenfrom hydrogen and Cl. In some examples of Formula B, R^(b) is chosenfrom substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted carboxyl. In some examples of Formula B, R^(b) is chosenfrom CH₂CO₂CH₂CH₃, CH₂CO₂Bu (where Bu indicates a butyl group), CH₂CO₂H,CH₂Ph (where Ph indicates a phenyl group), Ph, cyclohexane, andn-octane.

In some examples for Formula B, where R^(a) comprises H, the compoundsare of Formula B-1:

wherein R^(b) is chosen from substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, and substituted or unsubstituted carboxyl; and (+), (−), and (±)isomers, and pharmaceutically acceptable salts and prodrugs thereof.

In some examples of Formula B-1, R^(b) is chosen from CH₂CO₂CH₂CH₃,CH₂CO₂Bu (where Bu indicates a butyl group, such as n-butyl, isobutyl,sec-butyl, or tert-butyl), CH₂CO₂H, CH₂Ph (where Ph indicates a phenylgroup), Ph, cyclohexane, and n-octane.

In some examples for Formula B, where R^(a) is Cl, the compounds are ofFormula B-2:

wherein R^(b) is chosen from substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, and substituted or unsubstituted carboxyl; and (+), (−), and (±)isomers, and pharmaceutically acceptable salts and prodrugs thereof.

In some examples of Formula B-2, R^(b) is chosen from CH₂CO₂CH₂CH₃,CH₂CO₂Bu (where Bu indicates a butyl group), CH₂CO₂H, CH₂Ph (where Phindicates a phenyl group), Ph, cyclohexane, and n-octane.

In some examples for Formula B, where R^(a) is Cl and R^(b) isCH₂CO₂CH₂CH₃, the compounds are of Formula B-3:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is Cl and R^(b) is CH₂CO₂Bu,the compounds are of Formula B-4:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples of Formula B-4, the butyl group comprises a tert-butylgroup.

In some examples for Formula B, where R^(a) is Cl and R^(b) is CH₂CO₂H,the compounds are of Formula B-5:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is H and R^(b) isCH₂CO₂CH₂CH₃, the compounds are of Formula B-6:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is H and R^(b) is CH₂CO₂Bu,the compounds are of Formula B-7:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B-7, the butyl group comprises a tert-butylgroup.

In some examples for Formula B, where R^(a) is H and R^(b) is CH₂CO₂H,the compounds are of Formula B-8:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is H and R^(b) is CH₂Ph, thecompounds are of Formula B-9:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is H and R^(b) is Ph, thecompounds are of Formula B-10:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is H and R^(b) iscyclohexane, the compounds are of Formula B-11:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

In some examples for Formula B, where R^(a) is H and R^(b) is n-octane,the compounds are of Formula B-12:

and (+), (−), and (±) isomers, and pharmaceutically acceptable salts andprodrugs thereof.

Also disclosed are compounds having Formula VI

-   wherein R⁶, R⁷, R¹³, and R¹⁴ are independently chosen from hydrogen,    halogen, and hydroxyl;-   R⁵ and R¹² are independently chosen from hydrogen, halogen, and    substituted or unsubstituted alkyl;-   R²⁰ and R²¹ are independently chosen from SR²² or SO₂R²², where R²²    is chosen from substituted or unsubstituted alkyl, substituted or    unsubstituted amino, substituted or unsubstituted cycloalkyl,    substituted or unsubstituted heteroalkyl, substituted or    unsubstituted heterocycloalkyl, substituted or unsubstituted    alkenyl, substituted or unsubstituted cycloalkenyl, substituted or    unsubstituted heteroalkenyl, substituted or unsubstituted    heterocycloalkenyl, substituted or unsubstituted alkynyl,    substituted or unsubstituted heteroalkynyl, substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted alkoxyl, substituted or unsubstituted    aryloxyl, and substituted or unsubstituted carboxyl;-   and (+), (−), and (±) isomers, and pharmaceutically acceptable salts    and prodrugs thereof.

In specific examples of Formula VI, one or both R²⁰ and R²¹ areSO₂-alkyl (e.g., methyl) substituted with CO₂H, CO₂alkyl, phenyl, ormethoxylphenyl. In other examples of Formula VI, one or both R²⁰ and R²¹are S-alkyl (e.g., methyl) substituted with CO₂H, CO₂alkyl, phenyl, ormethoxylphenyl.

Pharmaceutical Compositions

The compounds described herein or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of the compound described herein or derivatives thereofin combination with a pharmaceutically acceptable carrier and, inaddition, can include other medicinal agents, pharmaceutical agents,carriers, or diluents. By pharmaceutically acceptable is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected compound withoutcausing unacceptable biological effects or interacting in a deleteriousmanner with the other components of the pharmaceutical composition inwhich it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of physiologically acceptable carriers include saline,glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, andbuffers with other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™ (ICI, Inc.; Bridgewater, N.J.), polyethyleneglycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.). To provide forthe administration of such dosages for the desired therapeutictreatment, compositions disclosed herein can advantageously comprisebetween about 0.1% and 99% by weight of the total of one or more of thesubject compounds based on the weight of the total composition includingcarrier or diluent.

Compositions containing the compound described herein or derivativesthereof suitable for parenteral injection can comprise physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

These compositions can also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be promoted by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Isotonic agents, for example, sugars, sodium chloride, and thelike can also be included. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds describedherein or derivatives thereof include capsules, tablets, pills, powders,and granules. In such solid dosage forms, the compounds described hereinor derivatives thereof is admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They can contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients. The disclosed compounds can also beincorporated into polymers, examples of which include poly (D-Llactide-co-glycolide) polymer for intracranial tumors;poly[bis(pcarboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio(as used in GLIADEL); chondroitin; chitin; and chitosan.

Liquid dosage forms for oral administration of the compounds describedherein or derivatives thereof include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms can contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, can contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions of the compounds described herein or derivatives thereoffor rectal administrations are optionally suppositories, which can beprepared by mixing the compounds with suitable non-irritating excipientsor carriers such as cocoa butter, polyethyleneglycol or a suppositorywax, which are solid at ordinary temperatures but liquid at bodytemperature and therefore, melt in the rectum or vaginal cavity andrelease the active component.

Dosage forms for topical administration of the compounds describedherein or derivatives thereof include ointments, powders, sprays, andinhalants. The compounds described herein or derivatives thereof areadmixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as can berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of the compositions.

The compositions can include one or more of the compounds describedherein and a pharmaceutically acceptable carrier. As used herein, theterm pharmaceutically acceptable salt refers to those salts of thecompound described herein or derivatives thereof that are, within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds described herein. The term saltsrefers to the relatively non-toxic, inorganic and organic acid additionsalts of the compounds described herein. These salts can be prepared insitu during the isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonatesalts, and the like. These can include cations based on the alkali andalkaline earth metals, such as sodium, lithium, potassium, calcium,magnesium, and the like, as well as non-toxic ammonium, quaternaryammonium, and amine cations including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. (See S. M.Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated hereinby reference in its entirety, at least, for compositions taught herein.)

Administration of the compounds and compositions described herein orpharmaceutically acceptable salts thereof to a subject can be carriedout using therapeutically effective amounts of the compounds andcompositions described herein or pharmaceutically acceptable saltsthereof as described herein for periods of time effective to treat adisorder.

The effective amount of the compounds and compositions described hereinor pharmaceutically acceptable salts thereof as described herein can bedetermined by one of ordinary skill in the art and includes exemplarydosage amounts for a mammal of from about 0.5 to about 200 mg/kg of bodyweight of active compound per day, which can be administered in a singledose or in the form of individual divided doses, such as from 1 to 4times per day. Alternatively, the dosage amount can be from about 0.5 toabout 150 mg/kg of body weight of active compound per day, about 0.5 to100 mg/kg of body weight of active compound per day, about 0.5 to about75 mg/kg of body weight of active compound per day, about 0.5 to about50 mg/kg of body weight of active compound per day, about 0.5 to about25 mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about 10mg/kg of body weight of active compound per day, about 20 mg/kg of bodyweight of active compound per day, about 10 mg/kg of body weight ofactive compound per day, or about 5 mg/kg of body weight of activecompound per day. The expression effective amount, when used to describean amount of compound in a method, refers to the amount of a compoundthat achieves the desired pharmacological effect or other effect, forexample an amount that results in enzyme inhibition.

Those of skill in the art will understand that the specific dose leveland frequency of dosage for any particular subject can be varied andwill depend upon a variety of factors, including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the species, age, body weight, general health, sex anddiet of the subject, the mode and time of administration, rate ofexcretion, drug combination, and severity of the particular condition.

Methods of Making the Compounds

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions can vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on the compounds discussed herein include the addition,subtraction, or movement of the various constituents as described foreach compound. Similarly, when one or more chiral centers are present ina molecule, the chirality of the molecule can be changed. Additionally,compound synthesis can involve the protection and deprotection ofvarious chemical groups. The use of protection and deprotection, and theselection of appropriate protecting groups can be determined by oneskilled in the art. The chemistry of protecting groups can be found, forexample, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference inits entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.),Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline(Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson(New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca(Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison,N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel,Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.),Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim (Ingelheim,Germany), or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Activity Assays

The activity of the compounds provided herein as anticancer agents canbe measured in standard assays, e.g., HPLC assays. The activities of thecompounds as determined using the assays described herein can bereported in terms of IC₅₀. As used herein, IC₅₀ refers to an amount,concentration, or dosage of a particular test compound that achieves a50% inhibition of a maximal response in an assay that measures suchresponse.

In certain aspects, the disclosed compounds and compositions need notactually be synthesized, but instead can be used as targets for anymolecular modeling technique to predict and characterize interactionswith cancer associated enzymes. This is achieved through structuralinformation and computer modeling. Computer modeling technology allowsvisualization of the three-dimensional atomic structure of a selectedmolecule and the rational design of new compounds that will interactwith an enzyme. The three-dimensional construct of the enzyme typicallydepends on data from x-ray crystallographic analyses or NMR imaging ofthe selected molecule. The molecular dynamics require force field data(e.g., Merck Molecular Force Field). The computer graphics systemsenable prediction of how a new compound will link to the enzyme andallow experimental manipulation of the structures of the compound toperfect binding specificity. Prediction of what the interactions will bewhen small changes are made in one or both requires molecular mechanicssoftware and computationally intensive computers, usually coupled withuser-friendly, menu-driven interfaces between the molecular designprogram and the user.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other. Uponidentification of compounds that interact in a desired way with theenzyme in silico, actual compounds can be synthesized and assayed asdisclosed herein.

The activity of the compounds provided herein as antibacterial agentscan be measured in standard assays, e.g., HPLC assays. The compounds canalso be evaluated for antibacterial activity using the Mueller Hinton(MH) broth antibacterial assay as specified by the Clinical andLaboratory Standards Institute MIC broth microdilution protocol (seeMethods for Dilution Antimicrobial Susceptibility Tests for BacteriaThat Grow Aerobically; Approved Standard, In The Clinical and LaboratoryStandards Institute (CLSI, formerly NCCLS), 7^(th) ed., January 2006, 26(2), M7-A7; see also Performance Standards for AntimicrobialSusceptibility Testing; Eighteenth Informational Supplement, In TheClinical and Laboratory Standards Institute (CLSI, formerly NCCLS),January 2008, 28 (1), M100-S18.

The activities of the compounds as determined using the assays describedherein can be reported in terms of IC₅₀ and/or MIC 100. As used herein,IC₅₀ refers to an amount, concentration or dosage of a particular testcompound that achieves a 50% inhibition of a maximal response in anassay that measures such response. MIC 100 is used to measure the growthinhibition of cells and refers to a 100% inhibition of cell growth.

In certain aspects, the disclosed compounds and compositions need notactually be synthesized, but instead can be used as targets for anymolecular modeling technique to predict and characterize interactionswith bacterial enzymes. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with a bacterialenzyme. The three-dimensional construct of the enzyme typically dependson data from x-ray crystallographic analyses or NMR imaging of theselected molecule. The molecular dynamics require force field data(e.g., Merck Molecular Force Field). The computer graphics systemsenable prediction of how a new compound will link to the enzyme andallow experimental manipulation of the structures of the compound toperfect binding specificity. Prediction of what the interactions will bewhen small changes are made in one or both requires molecular mechanicssoftware and computationally intensive computers, usually coupled withuser-friendly, menu-driven interfaces between the molecular designprogram and the user.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other. Uponidentification of compounds that interact in a desired way with thebacterial enzyme in silico, actual compounds can be synthesized andassayed as disclosed herein.

Methods of Use

Marinopyrroles were reported to show antibiotic activity againstmethicillin-resistant Staphylococcus aureus (MRSA) in 2008 by theFenical group (Hughes C C et al. Org. Lett. 2008, 10, 629-631). Due totheir molecular structures and promising biological properties,marinopyrroles have attracted considerable attention (Hughes C C et al.J. Org. Chem. 2010, 75, 3240-3250; Cheng C et al. J. Comb. Chem. 2010,12, 541-547; Kanakis A A and Sarli V. Org. Lett. 2010, 12, 4872-4875;Nicolaou K C et al. Tetrahedron Lett. 2011, 52, 2041-2043; Liu Y et al.Mar. Drugs 2012, 10, 953-962; Pan L et al. Chem. J. Chinese Universities2012, 33, 1476-1480; Yamanaka K et al. J. Am. Chem. Soc. 2012, 134,12434-12437; Cheng P et al. Chem. Commun. 2013, 49, 558-560; Clive D L Jand Cheng, P. Tetrahedron 2013, 69, 5067-5078; Cheng C et al. Mar. Drugs2013, 11, 2927-2948). The total synthesis of (±)-marinopyrrole A (1) wasreported along with 12 derivatives in early 2010 (Cheng C et al. J.Comb. Chem. 2010, 12, 541-547). This was soon followed by a report ofthe synthesis of (±)-marinopyrrole A via an intermolecular Ullmancoupling to form the bispyrrole system (Kanakis A A and Sarli V. Org.Lett. 2010, 12, 4872-4875). A five-step method to access marinopyrrolederivatives, (+)-1 and (−)-1 atropisomers after a chiral separation of(±)-1 using HPLC, as well as their antibiotic activities against MRSAwas reported in 2011 (Nicolaou K C et al. Tetrahedron Lett. 2011, 52,2041-2043). Afterwards, a biosynthetic approach toward marinopyrrole Avia an N, C-bispyrrole homocoupling catalyzed by two flavin-dependenthalogenases was described (Yamanaka K et al. J. Am. Chem. Soc. 2012,134, 12434-12437). More recently, the total synthesis of marinopyrrole Band a review of the marinopyrroles were reported (Cheng P et al. Chem.Commun. 2013, 49, 558-560; Clive D L J and Cheng, P. Tetrahedron 2013,69, 5067-5078). After the synthesis of a series of “non-symmetrical”marinopyrrole derivatives and their antibiotic activities was reported(Liu Y et al. Mar. Drugs 2012, 10, 953-962), optimization studies of thesteps to avoid the formation of oxazepine byproduct was reported (Pan Let al. Chem. J. Chinese Universities 2012, 33, 1476-1480). Mostrecently, potent marinopyrrole derivatives against MRSA were reported(Cheng C et al. Mar. Drugs 2013, 11, 2927-2948). Furthermore, it wasalso reported was that (±)-marinopyrrole A antagonizes Mcl-1 andovercomes resistance of human cancer cells to the Bcl-x_(L) antagonistABT-737 (Doi K et al. J. Biol. Chem. 2012, 287, 10224-10235). Aselective Mcl-1 small-molecule inhibitor blocking pancreatic cancergrowth in vitro and in vivo resulted from high throughput screeningfollowed by structure-based chemical optimization was reported recently(Abulwerdi F et al. Mol. Cancer Ther. 2013, published OnlineFirst Sep.9, 2013. doi: 10.1158/1535-7163.MCT-12-0767).

Provided herein are methods of treating, preventing, or amelioratingcancer in a subject. The methods include administering to a subject aneffective amount of one or more of the compounds or compositionsdescribed herein, or a pharmaceutically acceptable salt thereof. Thecompounds and compositions described herein or pharmaceuticallyacceptable salts thereof are useful for treating cancer in humans, e.g.,pediatric and geriatric populations, and in animals, e.g., veterinaryapplications. The disclosed methods can optionally include identifying apatient who is or can be in need of treatment of a cancer. Examples ofcancer types treatable by the compounds and compositions describedherein include bladder cancer, brain cancer, breast cancer, colorectalcancer, cervical cancer, gastrointestinal cancer, genitourinary cancer,head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, renal cancer, skin cancer, and testicular cancer.Further examples include cancer and/or tumors of the anus, bile duct,bone, bone marrow, bowel (including colon and rectum), eye, gallbladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix,mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina,vulva, uterus, liver, muscle, blood cells (including lymphocytes andother immune system cells). Some examples of cancers contemplated fortreatment include carcinomas, Karposi's sarcoma, melanoma, mesothelioma,soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acutelymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, andother), and lymphoma (Hodgkin's and non-Hodgkin's), and multiplemyeloma.

The methods of treatment or prevention described herein can furtherinclude treatment with one or more additional agents (e.g., ananti-cancer agent or ionizing radiation). The one or more additionalagents and the compounds and compositions or pharmaceutically acceptablesalts thereof as described herein can be administered in any order,including simultaneous administration, as well as temporally spacedorder of up to several days apart. The methods can also include morethan a single administration of the one or more additional agents and/orthe compounds and compositions or pharmaceutically acceptable saltsthereof as described herein. The administration of the one or moreadditional agents and the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein can be by the same ordifferent routes. When treating with one or more additional agents, thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein can be combined into a pharmaceutical compositionthat includes the one or more additional agents.

For example, the compounds or compositions or pharmaceuticallyacceptable salts thereof as described herein can be combined into apharmaceutical composition with an additional anti-cancer agent, such as13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA,2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine,Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort,Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran,All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole,Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenictrioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab,Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib,Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar,Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine,Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab,Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone,Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabineliposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetinalfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicinliposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukindiftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasonesodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome,Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin,Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol,Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane,Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara,Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron,Lupron Depot, Matulane, Maxidex, Mechlorethamine, -MechlorethamineHydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate,Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, MethotrexateSodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta,Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex,Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak,Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel,Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon,Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase,Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone,Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 withCarmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan,Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycinhydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef,Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol,Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide,Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa,Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulatingfactor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine,HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisonesodium phosphate, Hydrocortisone sodium succinate, Hydrocortonephosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate,Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEGconjugate), Interleukin 2, Interleukin-11, Intron A (interferonalfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine,Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin,Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX,Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan,Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. Theadditional anti-cancer agent can also include biopharmaceuticals suchas, for example, antibodies.

Further, the additional agent can include ABT-263 (CAS#923564-51-6) alsoknown as navitoclax and/or ABT-737 (CAS#852808-04-9), both of which arecommercially. Still further the disclosed compositions can furtherinclude compounds that inhibit transcription of Mcl-1, such as with thecyclin-dependent kinase inhibitors Seliciclib (CAS#186692-46-6) andFlavopiridol (CAS#146426-40-6) or translation, such as with themultikinase inhibitor BAY 43-9006 (CAS#284461-73-0). Further examples ofadditional compounds that can be present in the disclosed compositionsinclude, but are not limited to, dexamethasone (CAS#50-02-2), melphalan(CAS#148-82-3), obatoclax (CAS#803712-67-6), BH3-M6, and gossypol(CAS#303-45-7).

Many tumors and cancers have viral genome present in the tumor or cancercells. For example, Epstein-Barr Virus (EBV) is associated with a numberof mammalian malignancies. The compounds disclosed herein can also beused alone or in combination with anticancer or antiviral agents, suchas ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treatpatients infected with a virus that can cause cellular transformationand/or to treat patients having a tumor or cancer that is associatedwith the presence of viral genome in the cells. The compounds disclosedherein can also be used in combination with viral based treatments ofoncologic disease. For example, the compounds can be used with mutantherpes simplex virus in the treatment of non-small cell lung cancer(Toyoizumi, et al., “Combined therapy with chemotherapeutic agents andherpes simplex virus type IICP34.5 mutant (HSV-1716) in human non-smallcell lung cancer,” Human Gene Therapy, 1999, 10(18):17).

Also described herein are methods of killing a tumor cell in a subject.The method includes contacting the tumor cell with an effective amountof a compound or composition as described herein, and optionallyincludes the step of irradiating the tumor cell with an effective amountof ionizing radiation. Additionally, methods of radiotherapy of tumorsare provided herein. The methods include contacting the tumor cell withan effective amount of a compound or composition as described herein,and irradiating the tumor with an effective amount of ionizingradiation. As used herein, the term ionizing radiation refers toradiation comprising particles or photons that have sufficient energy orcan produce sufficient energy via nuclear interactions to produceionization. An example of ionizing radiation is x-radiation. Aneffective amount of ionizing radiation refers to a dose of ionizingradiation that produces an increase in cell damage or death whenadministered in combination with the compounds described herein. Theionizing radiation can be delivered according to methods as known in theart, including administering radiolabeled antibodies and radioisotopes.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. As used herein the term treatingor treatment includes prevention; delay in onset; diminution,eradication, or delay in exacerbation of signs or symptoms after onset;and prevention of relapse. For prophylactic use, a therapeuticallyeffective amount of the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein are administered to asubject prior to onset (e.g., before obvious signs of cancer), duringearly onset (e.g., upon initial signs and symptoms of cancer), or afteran established development of cancer. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. Prophylactic administration can be used, for example,in the chemopreventative treatment of subjects presenting precancerouslesions, those diagnosed with early stage malignancies, and forsubgroups with susceptibilities (e.g., family, racial, and/oroccupational) to particular cancers. Therapeutic treatment involvesadministering to a subject a therapeutically effective amount of thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein after cancer is diagnosed.

In some examples, the compounds disclosed herein are effective atinhibiting Mcl-1. In some examples, the compounds disclosed herein canbe used for treating cancers where Mcl-1 is expressed or over expressed,and killing Mcl-1 dependent cells.

Also provided herein are methods to treat, prevent, or limit microbialinfections in a subject. The methods include administering to a subjectan effective amount of one or more of the compounds or compositionsdescribed herein, or a pharmaceutically acceptable salt thereof. Thecompounds and compositions described herein or pharmaceuticallyacceptable salts thereof are useful for treating microbial infectionsand cancer in humans, e.g., pediatric and geriatric populations, and inanimals, e.g., veterinary applications. Microbial infections include,for example, bacterial and fungal infections. Bacterial infectionsinclude infections caused by bacilli, cocci, spirochaetes, and vibriobacteria. In some examples, the microbial infection is a bacterialinfection (e.g., a Gram positive bacterial infection). In some examples,the bacterial infection is Staphylococcus infection, such as aStaphylococcus aureus. The compounds and compositions described hereinare useful in treating a variety of Staphylococcus aureus infections,including drug-resistant Staphylococcus aureus infections andbiofilm-associated Staphylococcus aureus infections. In someembodiments, the Staphylococcus aureus infection ismethocillin-resistant S. aureus (MRSA). For example, the MRSA can behospital-associated MRSA or community associated MRSA.

The methods of treatment or prevention described herein can furtherinclude treatment with one or more additional agents (e.g., anantibacterial agent). The one or more additional agents and thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein can be administered in any order, includingsimultaneous administration, as well as temporally spaced order of up toseveral days apart. The methods can also include more than a singleadministration of the one or more additional agents and/or the compoundsand compositions or pharmaceutically acceptable salts thereof asdescribed herein. The administration of the one or more additionalagents and the compounds and compositions or pharmaceutically acceptablesalts thereof as described herein can be by the same or differentroutes. When treating with one or more additional agents, the compoundsand compositions or pharmaceutically acceptable salts thereof asdescribed herein can be combined into a pharmaceutical composition thatincludes the one or more additional agents. For example, the compoundsor compositions or pharmaceutically acceptable salts thereof asdescribed herein can be combined into a pharmaceutical composition withan additional antibacterial agent, such as acedapsone; acetosulfonesodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil;amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacinsulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin;amphomycin; ampicillin; ampicillin sodium; apalcillin sodium; apramycin;aspartocin; astromicin sulfate; avilamycin; avoparcin; azithromycin;azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin;bacitracin methylene disalicylate; bacitracin zinc; bambermycins;benzoylpas calcium; berythromycin; betamicin sulfate; biapenem;biniramycin; biphenamine hydrochloride; bispyrithione magsulfex;butikacin; butirosin sulfate; capreomycin sulfate; carbadox;carbenicillin disodium; carbenicillin indanyl sodium; carbenicillinphenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor;cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium;cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium;cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol;cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium;cefonicid monosodium; cefonicid sodium; cefoperazone sodium; ceforanide;cefotaxime sodium; cefotetan; cefotetan disodium; cefotiamhydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizolesodium; cefpiramide; cefpiramide sodium; cefpirome sulfate; cefpodoximeproxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime;ceftibuten; ceftizoxime sodium; ceftriaxone sodium; cefuroxime;cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrilesodium; cephalexin; cephalexin hydrochloride; cephaloglycin;cephaloridine; cephalothin sodium; cephapirin sodium; cephradine;cetocycline hydrochloride; cetophenicol; chloramphenicol;chloramphenicol palmitate; chloramphenicol pantothenate complex;chloramphenicol sodium succinate; chlorhexidine phosphanilate;chloroxylenol; chlortetracycline bisulfate; chlortetracyclinehydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride;cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin;clindamycin hydrochloride; clindamycin palmitate hydrochloride;clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillinsodium; cloxyquin; colistimethate sodium; colistin sulfate; coumermycin;coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone;daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline;denofungin; diaveridine; dicloxacillin; dicloxacillin sodium;dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline;doxycycline calcium; doxycycline fosfatex; doxycycline hyclate; droxacinsodium; enoxacin; epicillin; epitetracycline hydrochloride;erythromycin; erythromycin acistrate; erythromycin estolate;erythromycin ethylsuccinate; erythromycin gluceptate; erythromycinlactobionate; erythromycin propionate; erythromycin stearate; ethambutolhydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine;flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin;furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid;gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin;hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole;isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin;levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin;lincomycin hydrochloride; lomefloxacin; Lomefloxacin hydrochloride;lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocyclinesulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem;methacycline; methacycline hydrochloride; methenamine; methenaminehippurate; methenamine mandelate; methicillin sodium; metioprim;metronidazole hydrochloride; metronidazole phosphate; mezlocillin;mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycinhydrochloride; monensin; monensin sodiumr; nafcillin sodium; nalidixatesodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate;neomycin sulfate; neomycin undecylenate; netilmicin sulfate;neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone;nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole;nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium;ofloxacin; onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonamsodium; oxolinic acid; oxytetracycline; oxytetracycline calcium;oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin;pefloxacin; pefloxacin mesylate; penamecillin; penicillin G benzathine;penicillin G potassium; penicillin G procaine; penicillin G sodium;penicillin V; penicillin V benzathine; penicillin V hydrabamine;penicillin V potassium; pentizidone sodium; phenyl aminosalicylate;piperacillin sodium; pirbenicillin sodium; piridicillin sodium;pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillinpamoate; pivampicillin probenate; polymyxin B sulfate; porfiromycin;propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate;quinupristin; racephenicol; ramoplanin; ranimycin; relomycin;repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin;rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate;rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicinsodium phosphate; rosaramicin stearate; rosoxacin; roxarsone;roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin;sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin;spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride;steffimycin; streptomycin sulfate; streptonicozid; sulfabenz;sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine;sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene;sulfamerazine; sulfameter; sulfamethazine; sulfamethizole;sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc;sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet;sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine;sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillinhydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin;tetracycline; tetracycline hydrochloride; tetracycline phosphatecomplex; tetroxoprim; thiamphenicol; thiphencillin potassium;ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium;ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate;tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines;troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin;vancomycin hydrochloride; virginiamycin; or zorbamycin.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. As used herein the term treatingor treatment includes prevention; delay in onset; diminution,eradication, or delay in exacerbation of signs or symptoms after onset;and prevention of relapse. For prophylactic use, a therapeuticallyeffective amount of the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein are administered to asubject prior to onset (e.g., before obvious signs of a bacterialinfection), during early onset (e.g., upon initial signs and symptoms ofa bacterial infection), or after an established inflammatory response ordevelopment of a bacterial infection. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. Prophylactic administration can be used, for example,in the preventative treatment of subjects exposed to Staphylococcusaureus. Therapeutic treatment involves administering to a subject atherapeutically effective amount of the compounds and compositions orpharmaceutically acceptable salts thereof as described herein after abacterial infection is diagnosed.

Kits

Also provided herein are kits for treating or preventing cancer in asubject. A kit can include any of the compounds or compositionsdescribed herein. A kit can further include one or more anti-canceragents (e.g., paclitaxel). A kit can include an oral formulation of anyof the compounds or compositions described herein. A kit canadditionally include directions for use of the kit (e.g., instructionsfor treating a subject).

Also provided herein are kits for treating or preventing a bacterialinfection in a subject. A kit can include any of the compounds orcompositions described herein. A kit can further include one or moreantibacterial agents (e.g., oxacillin). A kit can include an oralformulation of any of the compounds or compositions described herein. Akit can additionally include directions for use of the kit (e.g.,instructions for treating a subject).

The examples below are intended to further illustrate certain aspects ofthe methods and compounds described herein, and are not intended tolimit the scope of the claims.

EXAMPLES

The following examples are set forth below to illustrate the methods,compositions, and results according to the disclosed subject matter.These examples are not intended to be inclusive of all aspects of thesubject matter disclosed herein, but rather to illustrate representativemethods, compositions, and results. These examples are not intended toexclude equivalents and variations of the present invention, which areapparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures, and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

All chemicals were purchased from commercial suppliers and used withoutfurther purification. All solvents were dried and distilled before use.Tetrahydrofuran was distilled from sodium/benzophenone. Dichloromethaneand acetonitrile were distilled over calcium hydride. Flash columnchromatography was performed with silica gel (200-300 mesh). ¹H NMRspectra were recorded at either 400 MHz or 600 MHz at ambienttemperature. ¹³C NMR spectra were recorded at either 100 or 150 MHz atambient temperature. Infrared spectra were recorded on aspectrophotometer (Perkin-Elmer Spectrum 100). Melting points weredetermined with melting point apparatus (Fukai X-4). High resolutionmass spectra were performed by electrospray ionization (ESI) on anAgilent ESI-TOF LC-MS 6200 system. Analytical HPLC was performed on anAgilent 1100 series with diode array detectors and auto samplers. Alltested compounds possessed a purity of not less than 95%.

Example 1

Previous reports have discussed the ability of marinopyrrole A toinhibit the binding of Mcl-1 to Bim. However, marinopyrrole A onlymoderately inhibits the binding to Mcl-1 to Bim (8.9 μM), itsselectivity for Mcl-1 over Bcl-xL is only two fold, and it suffers frompoor solubility. Herein, new marinopyrrole A analogues were synthesizedwhich can: have improved solubility and potency, identify chemicalprobes selective for Mcl-1, Bcl-xL or Bcl-2, and be developed asanti-cancer drugs. The molecular geometry of marinopyrrole A offersexcellent opportunities to reach these goals by decorating this naturalproduct based bispyrrole system with a large number of diversefunctional groups. Marinopyrrole has at least eight sites amenable tooptimization to accomplish the desired activity and selectivity.

Structure activity relationship studies using Mcl-1/Bim and Bcl-xL/BimELISA assays were used to identify marinopyrrole A analogues that areMcl-1-selective and Bcl-xL-selective antagonists as well as dualinhibitors of Bim binding to both Mcl-1 and Bcl-xL. The parentmarinopyrrole A [1=(±)−(1)] inhibits the binding of Mcl-1 to Bim withonly moderate potency (IC₅₀=8.9 μM) (Table 1). The potency of 1 atinhibiting Bcl-xL/Bim binding had an IC₅₀ of 16.4 μM. All ELISA datareported herein were performed with both Mcl-1 and Bcl-xL at 10 nM.

TABLE 1 ELISA results of selected marinopyroles ID R^(17e) Mcl-1-Bim^(a)Bcl-xL-Bim^(a) (±)-1 —  8.9 ± 1.0 16.4 ± 3.3 (+)-1 — 12.7 ± 1.0 19.7 ±3.6 (−)-1 — 12.5 ± 1.4 12.0 ± 2.8 IV-1 OSO₂CF₃  1.4 ± 0.3  2.3 ± 1.1IV-2 OH 42.5 ± 6.0 >100 IV-3 CO₂CH₃  4.3 ± 1.5  3.4 ± 0.9 IV-4 CO₂H 66.6± 2.6 >100 IV-5 PO(OCH₂CH₃)₂ >100 >100 IV-6 PO(OH)₂ >100 >100 ^(a)IC₅₀(average ± SEM) in μM, n ≧ 3.

Example 2

Marinopyrrole F (FIG. 1) was chosen as a starting point for optimization(Hughes C C et al. J. Org. Chem. 2010, 75, 3240-3250). Marinopyrrole Fadopted specific conformations that locked one aromatic ring to thebispyrrole system due to the fused eight-membered ether linkage as shownby crystallographic X-ray analysis (Hughes C C et al. J. Org. Chem.2010, 75, 3240-3250). Introduction of substituents in the para-positionrelative to the carbonyl group on both aromatic rings such astrifluoromethanesulfonate 3, methyl ester 4 or diethyl phosphonate 5functionality generated a series of compounds that can have hydrogenbonding (acceptor) and hydrophobic interactions with the target.Furthermore, the unmasked hydroxyl 6, carboxylic acid 7 and phosphonicacid 8 groups in the corresponding positions can serve as both ahydrogen bond donor/acceptor and a functional group to improve aqueoussolubility. To evaluate the potential differences in potency between theatropisomers of 1, both (+)-1 and (−)-1 marinopyrrole A were included inthis study (Nicolaou K C et al. Tetrahedron Lett. 2011, 52, 2041-2043).The biological activity of brominated marinopyrrole A analog 9 wasevaluated by ELISA assays (Nicolaou K C et al. Tetrahedron Lett. 2011,52, 2041-2043).

Synthesis of Marinopyrrole Derivatives

Starting from compound 2 (Cheng C et al. Mar. Drugs 2013, 11,2927-2948), macrocycle 3 was obtained in 80% yield after heating 2 inDMF at 110° C. (Scheme 2). Removal of the trifluoromethanesulfonicgroups by saponification in methanolic THF gave phenol 6 in 81% yield.Palladium-mediated carbonylation (Uyanik M et al. Org. Lett. 2006, 8,5649-5652) of 2 provided symmetrical marinopyrrole 4a and cyclicmarinopyrrole 4 in 25% and 22% yield, respectively. Further heating ofcompound 4a at 80° C. generated 4, which can be due to spontaneouscyclization of 8-OH with 5′-Cl. Saponification of 4 and 4a yielded thecorresponding carboxylic acid derivatives 7 and 7a, respectively (Scheme3). Palladium-catalyzed phosphorylation (Petrakis K S and Nagabhushan TL. J. Am. Chem. Soc. 1987, 109, 2831-2833) of 2 with HPO(OEt)₂ furnisheda mixture of symmetrical marinopyrrole 5a in 43% yield as well ascyclized 5 in 54% yield (Scheme 4). Intramolecular cyclization of 5a canalso occur upon heating at 81-82° C. Finally, upon treatment withMe₃SiBr, 5 and 5a can be smoothly converted to the correspondingbisphosphonic acids 8 and 8a (Petrakis K S and Nagabhushan T L. J. Am.Chem. Soc. 1987, 109, 2831-2833).

Physicochemical Properties and SAR of the Marinopyrroles

Consistent with previous reports (Doi K et al. J. Biol. Chem. 2012, 287,10224-10235), the IC₅₀ value of racemic marinopyrrole A to disrupt thebinding of Mcl-1 to Bim was 8.9 μM. Although the activity of racemicmarinopyrrole A against Bcl-x_(L)/Bim binding was lower than reportedpreviously (Doi K et al. J. Biol. Chem. 2012, 287, 10224-10235), thisreflects the lower Bcl-x_(L) concentration (2.5 times lower) utilized inthe present assay. No significant activity difference was observedbetween atropisomers (+)-1 and (−)-1, as both exhibited similarpotencies against Mcl-1/Bim and Bcl-x_(L)/Bim (FIG. 2). Symmetricallypara-substituted marinopyrroles with a carboxy methyl ester 4a anddiethyl phosphonate 5a showed activity against Mcl-1/Bim but wereinactive against Bcl-x_(L)/Bim (IC₅₀>100 μM). Furthermore, substitutionin the para-position of the carbonyl group with carboxylic acid 7ashowed lower activity than 1 against Mcl-1/Bim and little activityagainst Bcl-x_(L)/Bim. Bisphosphonic acid marinopyrrole 8a was slightlyless potent than 1 against Mcl-1-Bim but not Bcl-x_(L)/Bim. Thebrominated marinopyrrole congener 9 (Nicolaou K C et al. TetrahedronLett. 2011, 52, 2041-2043) was two-fold more potent than 1 against bothMcl-1/Bim and Bcl-x_(L)/Bim.

Both pK_(a) and log p values were calculated using ChemAxon SoftwareVersion 5.12.3 (Dixon S L and Jurs P C. J. Comp. Chem. 1993, 14,1460-1467; Csizmadia F et al. J. Pharm. Sci. 1997, 86, 865-871). ThepK_(a) values of marinopyrrole A (1) were predicted to be 7.8 (pK_(a) 1)and 8.4 (pK_(a) 2), respectively (FIG. 2). As reported previously (ChengC et al. Mar. Drugs 2013, 11, 2927-2948), the difference in pK_(a)values for the hydroxyl group in ring A and ring B can be due to theaxially chiral environment. The pK_(a) values of 1 are 1.6-2.2 log unitslower than that of phenol (pK_(a)=9.98) (Liptak M D et al. J. Am. Chem.Soc. 2002, 124, 6421-6427). An equilibrium can exist between openconformations and closed conformations in 1, similar to those observedin a report of intramolecular hydrogen bonding (Kuhn B et al. J. Med.Chem. 2010, 53, 2601-2611). The Fenical group reported the X-raystructure of marinopyrrole B (3′-Br analogue of 1) that indicated thepreference for the formation of intramolecular hydrogen bonds betweenthe peri-hydroxyl and the carbonyl group (Hughes C C et al. Org. Lett.2008, 10, 629-631). These intramolecular hydrogen bond interactions cancontribute not only to an increase of the compound's acidity but also anincrease in its lipophilicity (Kuhn B et al. J. Med. Chem. 2010, 53,2601-2611). The calculated log p value of 1 was 5.6, which marginallyviolates the Rule of Five (RO5), drug-like properties formulated byLipinski (Lipinski C A et al. Adv. Drug Del, Rev. 2001, 46, 3-26). Thecalculated pK_(a) 1 and pK_(a) 2 values of marinopyrroles in FIG. 2range from 6.8 to 8.4. Compound 7a has pK_(a) 3 (3.8) and pK_(a) 4 (3.2)values due to the carboxylic acid, while 8a has a pK_(a) 3 (0.7-5.5) andpK_(a) 4 (1.0-5.8) range of values corresponding to the phosphonic acidfunctional group. Clog p values of both compounds 7a (4.6) and 8a (2.4)reside within the suggested range for drug-like compounds. Despite theimprovement in aqueous solubility of 7a and 8a over 1, both were foundto be less active against Mcl-1/Bim and Bcl-x_(L)/Bim, which can be dueto unfavorable ionic and/or hydrogen bond interactions with the targets.

Compared to the SARs of symmetrical marinopyrroles described (videsupra), cyclic marinopyrroles behaved similarly. Compounds containingfunctional groups with potential ionic and/or hydrogen bond interactions(6-8) reduced both anti Mcl-1/Bim and Bcl-x_(L)/Bim activity, as thecyclic marinopyrroles phosphonic acid 8 and ester 5 lack activityagainst both Mcl-1/Bim and Bcl-x_(L)/Bim (IC₅₀>100 μM in FIG. 3).Conversely, methyl ester 4 is two-fold more potent than 1 againstMcl-1/Bim and seven-fold more potent against Bcl-x_(L)/Bim. Thetrifluoromethanesulphonate 3 was the most potent cyclic marinopyrroleexamined, showing six- and seven-fold higher potency than 1 againstMcl-1/Bim and Bcl-x_(L)/Bim, respectively. Compound 4 has a Clog p valueof 4.7, while compound 3 has a Clog p value outside the advised range ofRO5. The Clog p for compound 5 was marginally higher than the range ofRO5, while the rest of compounds (6-8) have the Clog p values all withinthe recommended range for RO5. This series of cyclic marinopyrroles,which adopt constrained molecular geometries due to the locked ringsystem (Hughes C C et al. J. Org. Chem. 2010, 75, 3240-3250), displayedenhanced ability to disrupt the binding of Bim to Mcl-1 and Bcl-x_(L).

Activity in Intact Human Breast Cancer Cells

To determine if the marinopyrroles were active in intact cells, humanbreast cancer MDA-MB-468 cells were treated with the marinopyrrolederivatives (10 μM for 16 h). The cells were then processed for westernblotting according to a previously described methods (Balasis M E et al.Clin. Cancer Res. 2011, 17, 2852-2862). FIG. 4 shows that treatment ofthe cells with 4a resulted in a significant decrease in the levels ofMcl-1 and Bim, and cleavage of caspase 3. Compound 7a, the freecarboxylic acid analogue of 4a, did not decrease Mcl-1 and Bim, andresulted in little caspase 3 cleavage. The phosphate 8a and itscorresponding ethyl ester 5a had little effect on Mcl-1, Bim or caspase3 (FIG. 4). The (±)-marinopyrrole A (1) (Doi K et al. J. Biol. Chem.2012, 287, 10224-10235) and its atropisomers (+)-1 and (−)-1 as well as9, tetrabromo-(±)-1, were able to decrease Mcl-1 and Bim and to cleavecaspase 3. However, none of the cyclic marinopyrroles were active inintact cells.

Synthesis of Marinopyrrole Derivatives3-Hydroxy-4-(2,3,7-trichloro-13-oxo-10-(((trifluoromethyl)sulfonyl)oxy)-1,13dihydrobenzo[g]di-pyrrolo[2,1-b:3′,2′-d][1,3]oxazocine-5-carbonyl)phenyltrifluoromethanesulfonate (3)

Under N₂,(4,4′,5,5′-tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-hydroxytrifluoromethanesulfonate)-phenyl)methanone)(2) (Cheng C et al. Mar. Drugs 2013, 11, 2927-2948) (150 mg, 0.19 mmol)and NaI (120 mg, 0.75 mmol) were dissolved in DMF (5 mL). The mixturewas heated to 110° C. and stirred for about 24 h. The reaction wasquenched by addition of saturated aqueous Na₂S₂O₃ (20 mL) and extractedwith EtOAc (15 mL×3). The suspension was filtered and the filtrate wasconcentrated in vacuum. The residue was purified by flash columnchromatography (16% EtOAc/petroleum ether, R_(f)=0.2) to give 3 (115 mg,80%) as a light yellow solid. mp 94.8-96.4° C.; ¹H NMR (400 MHz, CDCl₃)δ 5.72 (s, 1H), 6.89 (dd, J=8.8, 2.0 Hz, 1H), 6.98 (d, J=2.4 Hz, 1H),7.39 (dd, J=8.8, 2.0 Hz, 1H), 7.73 (d, J=2.4 Hz, 1H), 7.92 (d, J=8.8 Hz,1H), 8.14 (d, J=8.8 Hz, 1H), 9.72 (br s, 1H), 11.55 (s, 1H) ppm; ¹³C NMR(CDCl₃, 100 MHz) δ 101.68, 106.22, 111.40, 112.60, 116.88, 117.00,119.02, 120.19, 120.98, 121.12, 121.28, 122.85, 124.18, 125.33, 129.73,134.45, 135.18, 144.90, 152.81, 154.62, 157.16, 164.28, 174.98, 186.66ppm; HRMS (M+H⁺) calcd for C₂₄H₁₀Cl₃F₆N₂O₁₀S₂ 768.8747. found 768.8809;IR (KBr) 3423, 3244, 2960, 2922, 2852, 1626, 1604, 1580, 1462, 1426,1217, 1139, 1095, 965, 790 cm⁻¹. HPLC purity, 99.1% (Flow rate: 1mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6 mm; Wavelength:UV 254 nm; Temperature: 25° C.; Mobile phase: MeOH:H₂O=80:20; t_(R)=28.9min).

2,3,7-Trichloro-5-(2,4-dihydroxybenzoyl)-10-hydroxybenzo[g]dipyrrolo[2,1-b:3′,2′-d][1,3]oxazo-cin-13(1H)-one(6)

To a solution of 3 (65 mg, 0.08 mmol) in a mixture of MeOH/THF (1:1, 4mL), KOH (47 mg, 0.80 mmol) was added at room temperature. The mixturewas heated to 70° C. and stirred for 10 h. The reaction mixture wasadjusted to pH 7.0 with 0.5 N HCl and extracted with EtOAc (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by flashcolumn chromatography (33% EtOAc/petroleum ether, R_(f)=0.3) to yield 6(34 mg, 81%) as a yellow solid. mp 274.7-276.0° C.; ¹H NMR (400 MHz,acetone-d₆) δ 6.37 (s, 1H), 6.84 (d, J=2.0 Hz, 1H), 6.95 (br s, 1H),7.44 (dd, J=8.8, 2.4 Hz, 1H), 7.72 (d, J=2.0 Hz, 1H), 8.17 (dd, J=8.4,3.2 Hz, 1H), 8.38 (d, J=8.4 Hz, 1H) ppm; ¹³C NMR (CD₃OD, 100 MHz) δ91.20, 94.31, 96.54, 100.56, 100.90, 105.20, 106.97, 109.25, 113.22,113.86, 115.03, 115.39, 115.68, 125.60, 125.62, 127.17, 150.97, 156.11,157.43, 158.23, 168.02 178.12 ppm; HRMS (M+K⁺) calcd for C₂₂H₁₁Cl₃KN₂O₆542.9320. found 542.9297; IR (KBr) 3415, 3251, 2962, 2924, 1619, 1581,1547, 1476, 1456, 1310, 1090, 796 cm⁻¹. HPLC purity, 98.9% (Flow rate: 1mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6 mm; Wavelength:UV 254 nm; Temperature: 25° C.; Mobile phase: MeOH:H₂O=75:25; t_(R)=7.5min).

Methyl-2,3,7-trichloro-5-(2-hydroxy-4-(methoxycarbonyl)benzol)-13-oxo-1,13-dihydrobenzo[g]-dipyrrolo[2,1-b:3′,2′-d][1,3]oxazocine-10-carboxylate(4) andDimethyl-4,4′-(4,4′,5,5′-tetrachloro-1′H-[1,3′-bipyrrole]-2,2′-dicarbonyl)bis(3-hydroxybenzoate)(4a)

Under CO (1 atm), 2 (400 mg, 0.50 mmol), DPPP (26 mg, 0.10 mmol),Pd(OAc)₂ (11 mg, 0.05 mmol) and Et₃N (251 mg, 2.50 mmol) were dissolvedin a mixture of DMF/MeOH (5:1, 5 mL). The reaction was heated to 80° C.and stirred for 3 h. The reaction mixture was quenched with water (10mL) and extracted with EtOAc (15 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by flash column chromatography (50%EtOAc/petroleum ether, R_(f)=0.2) to give 4 (70 mg, 22%) and 4a (80 mg,25%) as a pale yellow solid.

4: mp 135.7-137.0° C.; ¹H NMR (400 MHz, CDCl₃) δ 3.96 (s, 3H), 3.98 (s,3H), 5.72 (s, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.84 (d, J=6.8Hz, 1H), 8.07 (s, 2H), 8.40 (s, 1H), 9.88 (s, 1H), 11.22 (s, 1H) ppm;¹³C NMR (acetone-d₆, 100 MHz) δ 52.81, 53.16, 107.56, 118.58, 118.60,120.76, 124.61, 128.87, 132.99, 132.99, 133.71, 134.02, 134.26, 136.82,136.99, 149.00, 157.40, 157.51, 164.91, 165.33, 166.12, 167.90, 174.87,176.18, 178.01, 183.00 ppm; HRMS (M+H⁺) calcd for C₂₆H₁₆Cl₃N₂O₈588.9972. found 588.9967; IR (KBr) 3416, 3236, 2954, 2852, 1730, 1609,1580, 1461, 1414, 1288, 1207, 1090, 988, 806 cm⁻¹. HPLC purity, 95.6%(Flow rate: 1 mL/min; Column: Waters C18, 5 m, 150×4.6 mm; Wavelength:UV 254 nm; Temperature: 25° C.; Mobile phase: MeOH:H₂O=90:10; t_(R)=4.6min).

4a: mp 99.4-101.0° C.; ¹H NMR (400 MHz, acetone-d₆) δ 3.84 (s, 3H), 3.86(s, 3H), 6.18 (s, 1H), 7.27 (dd, J=8.4, 1.6 Hz, 1H), 7.42-7.44 (m, 3H),7.87 (d, J=8.4 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H) ppm; ¹³C NMR (acetone-d₆,100 MHz) δ 52.68, 52.75, 110.16, 118.28, 118.51, 119.94, 120.60, 122.61,123.20, 124.43, 126.43, 126.59, 127.10, 128.72, 130.72, 133.06, 135.43,136.51, 158.90, 159.95, 166.17, 166.17, 184.78, 185.08, 185.60, 186.13ppm; HRMS (M+H⁺) calcd for C₂₆H₁₇Cl₄N₂O₈ 624.9739. found 624.9736; IR(KBr) 3245, 2954, 1727, 1632, 1599, 1441, 1291, 1223, 1093, 884, 759,672 cm⁻¹. HPLC purity, 96.3% (Flow rate: 1 mL/min; Column: AgilentZORBAX 300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature:25° C.; Mobile phase: MeOH:H₂O=80:20; t_(R)=6.6 min).

5-(4-Carboxy-2-hydroxybenzoyl)-2,3,7-trichloro-13-oxo-1,13-dihydrobenzo[g]dipyrrolo[2,1-b:3′,2′-d][1,3]oxazocine-10-carboxylicacid (7)

To a solution of 4 (44 mg, 0.07 mmol) in a mixture of H₂O/THF (1:2, 5mL), LiOH (27 mg, 1.1 mmol) was added at room temperature. The reactionwas heated to 70° C. and stirred for 10 h. The reaction mixture wasadjusted to pH 5.0 with 0.5 N HCl and extracted with EtOAc (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified byreverse-phase flash column chromatography (6% AcOH, 23% H₂O, 71% MeOH,R_(f)=0.2) to give 7 (30 mg, 71%) as a light yellow solid. mp215.5-217.0° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 6.03 (s, 1H), 7.40 (m, 3H),8.03 (m, 2H), 8.14 (s, 1H) ppm; ¹³C NMR (DMSO-d₆, 100 MHz) δ 100.08,107.72, 117.52, 120.03, 120.62, 123.74, 124.50, 124.73, 125.18, 128.83,130.17, 130.78, 132.98, 133.43, 136.00, 139.29, 145.72, 156.81, 156.81,166.57, 167.44, 173.04, 175.98, 183.02 ppm; HRMS (M+H⁺) calcd forC₂₄H₁₂Cl₃N₂O₈ 560.9659. found 560.9669; IR (KBr) 3420, 3240, 3127, 2925,2600, 1710, 1604, 1580, 1462, 1413, 1311, 1210, 1025, 996, 906, 799, 761cm⁻¹. HPLC purity, 99.3% (Flow rate: 1 mL/min; Column: Waters C18, 5 m,150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=55:45; t_(R)=6.7 min).

4,4′-(4,4′,5,5′-Tetrachloro-1′H-[1,3′-bipyrrole]-2,2′-dicarbonyl)bis(3-hydroxybenzoicacid) (7a)

To a solution of 4a (27 mg, 0.04 mmol) in a mixture of H₂O/THF (1:2, 3mL), LiOH (16 mg, 0.65 mmol) was added at room temperature. The reactionwas heated to 70° C. and stirred for 10 h. The reaction mixture wasadjusted to pH 5.0 with 0.5 N HCl and extracted with EtOAc (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified byreverse-phase flash column chromatography (6% AcOH, 30% H₂O, 64% MeOH,R_(f)=0.2) to give 7a (19 mg, 74%) as a light yellow solid. mp190.5-192.0° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 6.10 (s, 1H), 7.19 (d,J=8.0 Hz, 2H), 7.29-7.33 (m, 4H) ppm; ¹³C NMR (DMSO-d₆, 100 MHz) δ109.69, 110.24, 116.80, 117.21, 118.60, 118.66, 120.03, 122.32, 122.57,124.96, 129.10, 129.26, 129.80, 129.90, 129.92, 134.57, 135.43, 156.09,156.47, 167.58, 167.58, 172.66, 181.88, 183.14 ppm; HRMS (M+Na⁺) calcdfor C₂₄H₁₂Cl₄N₂NaO₈ 618.9245. found 618.9258; IR (KBr) 3075, 2956, 2919,2851, 1707, 1631, 1599, 1446, 1394, 1294, 1228, 1023, 995, 885, 760cm⁻¹. HPLC purity, 98.6% (Flow rate: 1 mL/min; Column: Waters C18, 5 m,150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25 OC; Mobile phase:MeOH:H₂O=65:35; t_(R)=5.1 min).

3-Hydroxy-4-(2,3,7-trichloro-13-oxo-10-(diethylphosphonyl)-1,13-dihydrobenzo[g]dipyrrolo[2,1-b:3′,2′-d][1,3]oxazocine-5-carbonyl)diethylphosphonate (5) andtetraethyl((4,4′,5,5′-tetrachloro-1′H-[1,3′-bipyrrole]-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene))bis(phosphonate)(5a)

Under N₂, 2 (50 mg, 0.06 mmol), diethyl phosphonate (52 mg, 0.36 mmol),Pd(PPh₃)₄ (7.6 mg, 0.006 mmol) and i-Pr₂NEt (48 mg, 0.36 mmol) weredissolved in anhydrous MeCN (5 mL). The reaction was heated to refluxand stirred for 10 h. The reaction mixture was quenched with water (10mL) and extracted with EtOAc (15 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by flash column chromatography (50%EtOAc/petroleum ether, R_(f)=0.2) to give 5 (25 mg, 54%) and 5a (20 mg,43%) as a yellow solid. 5: mp 122.8-124.3° C.; ¹H NMR (400 MHz, CDCl₃) δ1.36 (t, J=6.8 Hz, 12H), 4.12-4.24 (m, 8H), 5.71 (s, 1H), 7.36 (t, J=8.4Hz, 1H), 7.47 (d, J=15.2 Hz, 1H), 7.87 (m, 2H), 8.08 (dd, J=7.6, 5.2 Hz,1H), 8.17 (d, J=13.6 Hz, 1H), 9.85 (br s, 1H), 11.27 (s, 1H) ppm; ¹³CNMR (CDCl₃, 100 MHz) δ 16.02, 16.02, 16.27, 16.27, 62.64, 62.70, 62.83,62.83, 101.22, 106.12, 121.00, 121.59, 121.74, 123.06, 124.32, 125.10,126.46, 130.33, 132.61, 132.77, 132.89, 135.07, 135.93, 136.94, 137.75,145.48, 156.50, 161.50, 175.82, 187.12 ppm; HRMS (M+H⁺) calcd forC₃₀H₃₀Cl₃N₂O₁₀P₂ 745.0441. found 745.0454; IR (KBr) 3421, 3338, 3123,3078, 2983, 2925, 2855, 1614, 1579, 1461, 1258, 1232, 1050, 1021, 796cm⁻¹. HPLC purity, 97.2% (Flow rate: 1 mL/min; Column: Agilent ZORBAX300SB-C8, 5 m, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=80:20; t_(R)=6.8 min).

5a: mp 100.7-101.5° C.; ¹H NMR (400 MHz, CDCl₃) δ 1.24-1.36 (m, 12H),3.98-4.22 (m, 8H), 6.15 (s, 1H), 6.91 (dd, J=11.6, 8.0 Hz, 1H),7.24-7.27 (m, 1H), 7.30 (d, J=14.4 Hz, 1H), 7.40 (d, J=14.8 Hz, 1H),7.52 (t, J=14.4 Hz, 1H), 7.56 (dd, J=7.6, 2.8 Hz, 1H), 8.00 (br s, 1H),11.12 (s, 1H), 11.44 (br s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 16.22,16.22, 16.28, 16.28, 62.69, 62.69, 62.74, 62.74, 108.90, 111.95, 117.68,120.73, 121.03, 121.38, 121.47, 121.59, 122.07, 122.65, 122.84, 124.79,130.95, 133.35, 135.84, 137.67, 160.29, 160.48, 161.32, 161.52, 185.51,187.50 ppm; HRMS (M+H⁺) calcd for C₃₀H₃₁Cl₄N₂O₁₀P₂ 781.0208. found781.0220; IR (KBr) 3416, 3214, 2964, 2926, 2867, 1631, 1449, 1406, 1259,1222, 1022, 938, 800, 671 cm⁻¹. HPLC purity, 97.0% (Flow rate, 1 mL/min;Column: Phenomenex C6-phenyl, 5 m, 150×4.6 mm; Wavelength: UV 254 nm;Temperature: 25° C.; Mobile phase: MeOH:H₂O=80:20; t_(R)=4.0 min).

3-Hydroxy-4-(2,3,7-trichloro-13-oxo-10-phosphoryl-1,13-dihydrobenzo[g]dipyrrolo[2,1-b:3′,2′-d][1,3]oxazocine-5-carbonyl)phosphonic acid (8)

To a solution of 5 (40 mg, 0.054 mmol) in MeCN (3 mL), Me₃SiBr (230 mg,1.50 mmol) was added via a syringe at room temperature under N₂. Thereaction was heated to reflux and stirred for 24 h. The reaction mixturewas concentrated in vacuum. The residue was purified by reverse-phaseflash column chromatography (6% AcOH, 47% H₂O, 47% MeOH, R_(f)=0.2) togive 8 (27 mg, 79%) as a yellow solid. mp 314.7-316.0° C.; ¹H NMR (400MHz, CD₃OD) δ 5.95 (s, 1H), 7.26 (dd, J=12.8, 8.4 Hz, 1H), 7.32 (d,J=14.8 Hz, 1H), 7.53 (dd, J=8.0, 4.4 Hz, 1H), 7.85 (dd, J=12.8, 8.0 Hz,1H), 8.08 (dd, J=8.0, 4.4 Hz, 1H), 8.15 (d, J=13.6 Hz, 1H) ppm; ¹³C NMR(CD₃OD, 100 MHz) δ 101.57, 101.69, 106.80, 107.56, 120.56, 121.47,122.50, 124.67, 125.16, 125.52, 125.87, 126.60, 127.10, 130.67, 132.50,133.46, 136.20, 141.71, 147.04, 162.26, 177.27, 186.48 ppm; HRMS (M+H⁺)calcd for C₂₂H₁₄Cl₃N₂O₁₀P₂ 632.9189. found 632.9193; IR (KBr) 3790,3407, 2955, 2920, 2850, 1727, 1596, 1458, 1401, 877 cm⁻¹. HPLC purity,99.7% (Flow rate: 1 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm,150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=55:45; t_(R)=4.1 min).

((4,4′,5,5′-Tetrachloro-1′H-[1,3′-bipyrrole]-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene))diphosphonicacid (8a)

To a solution of 5a (18 mg, 0.023 mmol) in MeCN (3 mL), Me₃SiBr (99 mg,0.65 mmol) was added via a syringe at room temperature under N₂. Thereaction was heated to reflux and stirred for 24 h. The reaction mixturewas concentrated in vacuum. The residue was purified by reverse-phaseflash column chromatography (6% AcOH, 30% H₂O, 64% MeOH, R_(f)=0.2) togive 8a (13 mg, 84%) as a yellow solid. mp 317.6-318.7° C.; ¹H NMR (400MHz, CD₃OD) δ 6.29 (s, 1H), 7.05 (s, 1H), 7.26-7.37 (m, 5H) ppm; ¹³C NMR(CD₃OD, 100 MHz) δ 110.32, 110.38, 112.63, 114.07, 118.96, 120.50,121.60, 122.41, 123.23, 123.83, 125.59, 125.94, 126.34, 127.43, 129.35,130.79, 132.80, 136.61, 159.27, 159.82, 185.99, 187.14 ppm; HRMS (M+H⁺)calcd for C₂₂H₁₅Cl₄N₂O₁₀P₂ 668.8956. found 668.8958; IR (KBr) 2955,2919, 2850, 1626, 1464, 1020, 799 cm⁻¹. HPLC purity, 99.5% (Flow rate: 1mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6 mm; Wavelength:UV 254 nm; Temperature: 25° C.; Mobile phase: MeOH:H₂O=55:45; t_(R)=4.0min).

Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting FollowingTreatment of Intact Human Breast Cancer Cells

ELISAs were performed using a modified version of a previously describedprocedure (Doi K et al. J. Biol. Chem. 2012, 287, 10224-10235). Briefly,40 nM of biotinylated Bim BH3 peptide (Biomatik) in SuperBlock blockingbuffer (Pierce) was incubated in high-binding capacitystreptavidin-coated plates (Pierce) for 2 h. Compounds were diluted in120 μl of PBS containing 10 nM of GST-Mcl-1 or GST-Bcl-x_(L) in 1.5 mLtubes for 15 min. Wells were washed with wash buffer (PBS containing0.05% Tween-20) and then 100 μL of the compound/GST-protein mixture wastransferred to the wells. The plates were incubated for 2 h, and thenthe wells were washed with wash buffer. HRP-conjugated anti-GST antibody(Bethyl Laboratories) was diluted 1:2000 in SuperBlock and 100 μL wastransferred to each well. The plate was incubated for 1 h, and then thewells were washed with wash buffer followed by PBS. 100 μL of SureBlueTMB Microwell Peroxidase Substrate (VWR) was added to each well andplates were developed for 5-10 min. 100 μL of 1 N HCl was added to eachwell to stop the reaction and absorbance was read at 450 nm using aQuant plate reader (Bio-Tek). Treatment of the human breast cancer(MDA-MB-468) cells and western blotting were performed using apreviously described method (Balasis M E et al. Clin. Cancer Res. 2011,17, 2852-2862).

Described herein are general synthetic routes to access cyclicmarinopyrrole derivatives and evaluation of their in vitro activityagainst binding of the pro-survival proteins Mcl-1 and Bcl-x_(L) to thepro-apoptotic protein Bim. The efforts were focused on improving antiMcl-1/Bim and Bcl-x_(L)/Bim potency. SAR studies of marinopyrrolederivatives demonstrated: i) replacing the chlorines with bromineswithin the bispyrrole core improved the potency by two fold (1 vs. 9);ii) symmetrical marinopyrroles with substituents in the para-position tothe carbonyl group are more potent against Mcl-1/Bim than Bcl-x_(L)/Bim(FIG. 2); iii) the same trend was observed for cyclic marinopyrroles(FIG. 3); iv) cyclic marinopyrrole 3 is six- and seven-fold more potentthan 1 against Mcl-1/Bim and Bcl-x_(L)/Bim, respectively (FIG. 3); v)the cyclic marinopyrroles with certain substituents (OSO₂SF₃ and CO₂Me)in the para-position to the carbonyl group are excellent “leads” forfurther optimization. The disclosed cyclic marinopyrroles have improvedpotency against both Mcl-1 and Bcl-x_(L).

Example 3

A series of marinopyrrole derivatives were designed and synthesized.Their activity to disrupt the binding of the pro-apoptotic protein Bimto the pro-survival proteins Mcl-1 and Bcl-x_(L) was evaluated usingELISA assays. Structural characterization of marinopyrroles binding toMcl-1 was performed using NMR chemical shift perturbations assisted withmolecular modeling. Analogues where the 4- and 4′-hydrogens of phenylrings A and B in the synthetic marinopyrrole A (±)-1, a racemic form ofthe parent natural product marinopyrrole A (−)-1, were replaced bysulfide- or bistriazole-containing moieties such as 1-10 and 1-23 werethe most potent [500 nM; up to 32-fold more potent than (±)-1]. The mostpotent dual Mcl-1 and Bcl-x_(L) antagonists were sulfide 1-10 andtriazole 1-21. Furthermore, bistriazole 1-21 display chemical shiftperturbations on G219, G230 and G271 of Mcl-1 suggesting that it bindsin to the BH3 binding domain of Mcl-1. Several Marinopyrrole derivativesinhibit human breast cancer cell survival potently suggesting that thesecompounds exhibit anticancer activity.

Design of Disruptors of Mcl-1/Bim and BclxL/Bim Protein-ProteinInteractions

The molecular geometry of marinopyrrole A (1) offers excellentopportunities to decorate this natural product-based bispyrrole systemfor desired activity and selectivity. Previous reports (Doi K, et al. JBiol. Chem. 2012, 287, 10224-10235) have shown, via docking studiesbased on NMR chemical shift perturbations, that marinopyrrole A binds toMcl-1 in two major regions. One is centered at the Mcl-1 p2 pocketbetween helices 4 and 5 and in contact with helix 3. The other iscentered at the p4 pocket between helices 5 and 8 and in contact withhelix 2. In order to have comprehensive understanding of SARs, potentialsites of marinopyrrole A amenable for optimization were identified(Table 2). A series of marinopyrrole derivatives with substitution atthe para-position of two phenyl rings to the carbonyl groups was focusedon. Di-substitutions with hydrophobic groups on both phenyl ringsfurnished compounds 1-1 to 1-6, while those with hydrophilic groupsyielded derivatives 1-7, 1-8, 1-15 and 1-16. Tri-substitutions of“symmetrical” marinopyrroles on both phenyl rings provided compounds1-2, 1-8, and 1-17 to 1-19. Design of “non-symmetrical” marinopyrrolesincluded compounds 1-27 to 1-33, 1-36 and 1-37. Extension of functionalgroups in the para-position of the phenyl rings with sulfide or sulphonespacer furnished compounds 1-9 to 1-16. Marinopyrroles with bistriazolespacer, compounds 1-17 to 1-26, were designed to assess the bindingalong the entire α-helix from p1 to p4 pockets. N-methyl analogues 1-34and 1-35 were designed to further assess the role of the free NH group.“Symmetrical” and “non-symmetrical” marinopyrroles have been furtherdiscussed in PCT/US2014/012442 and WO 2013/158197, respectively, whichare incorporated herein by reference.

Chemistry and Synthesis

Starting from previously reported compound 2 (Cheng C, et al. Mar.Drugs. 2013, 11, 2927-2948), mono-ketone 4 was obtained in 73% yieldover two steps by introduction of ortho-methoxy-para-methylphenyl group(3 was not isolated) followed by IBX oxidation (Scheme 5). Removal ofthe TBDMS protecting group with TBAF gave alcohol 5 in 90% yield.Oxidation of 5 by IBX furnished aldehyde 6 in 90% yield. Bisketone 8 wasobtained in 54% yield after introduction of a secondortho-methoxy-para-methylphenyl group (without isolation of 7) followedby IBX oxidation. Removal of the para-toluenesulfonyl group with KOHgenerated 9 in 98% yield, which was converted to 10 in 65% yield bychlorination with NCS (Cheng C, et al. Mar. Drugs. 2013, 11, 2927-2948).The symmetrical marinopyrrole derivative 1-2 was obtained in 85% yieldafter demethylation using BBr₃/DCM (Cheng C, et al. J. Comb. Chem. 2010,12, 541-547).

Using previously reported intermediate 11 (Cheng C, et al. Mar. Drugs.2013, 11, 2927-2948) as a starting material, palladium-mediatedsubstitution of the triflate 11 with ethynyltrimethylsilane furnished 12in 74% yield (Scheme 6). Demethylation of 12 using BBr₃/DCM gave 13 in53% yield, which was converted to the symmetrical marinopyrrole 1-3 in78% yield. Reduction of triple bonds in 1-3 with atmospheric H₂/Pd/BaSO₄provided para-vinyl substituted marinopyrrole 1-4 in 60% yield, whichwas further reduced with atmospheric H₂/Pd/BaSO₄ to para-ethylmarinopyrrole 1-5 in 96% yield.

As shown in Scheme 7, palladium-mediated nucleophilic substitution ofthe triflate 14 (Cheng C, et al. Mar. Drugs. 2013, 11, 2927-2948) withethyl 2-mecaptoacetate, phenylmethanethiol and(4-methoxyphenyl)methanethiol gave 1-9 (61%), 1-10 (96%) and 1-11 (85%),respectively. Sulfides 1-9, 1-10 and 1-11 were oxidized to thecorresponding sulfones 1-12 (70%), 1-13 (75%) and 1-14 (65%) withm-chloroperbenzoic acid (m-CPBA), respectively. The carboxylic acids1-15 and 1-16 were obtained by saponification of the correspondingesters 1-9 and 1-12 using LiOH in 85% and 95% yields, respectively.

Schemes 8-10 show the chemistry that was developed to synthesizebistriazole marinopyrrole derivatives. Starting from a commonintermediate 15 (Cheng C, et al. Mar. Drugs. 2013, 11, 2927-2948),palladium-mediated substitution of the triflate 15 withethynyltrimethylsilane provided 16 in 92% yield, which was converted to17 in 95% yield after removal of tosyl group by KOH. Bistriazolemarinopyrrole 18 in 78% yield was constructed using “Click Chemistry”(Kolb H C, et al. Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021).Chlorination of 18 with NCS generated 19. The final product 1-17 wasobtained in 50% yield after demethylation of 19 using BBr₃/DCM (Cheng C,et al. J. Comb. Chem. 2010, 12, 541-547).

In order to improve overall yield, demethylation of 15 (Cheng C, et al.Mar. Drugs. 2013, 11, 2927-2948) using BBr₃/DCM was performed first togive 20 in 90% yield, as shown in Scheme 9. Palladium-mediatedsubstitution of the triflate 20 with ethynyltrimethylsilane furnished 21in 98% yield. Removal of the tosyl group in 21 provided 22 in 95% yield.Intermediate 23 was obtained in 80% yield using “Click Chemistry” (KolbH C, et al. Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021), which wassubjected to chlorination with NCS to give the final compound 1-18. Thefree carboxylic acid 1-19 was obtained in 65% yield after removal ofbutyl group from 1-18.

Compound 1-3 was used as a common starting material by “Click Chemistry”(Kolb H C, et al. Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021) toproduce seven bistriazole marinopyrrole derivatives (1-20-1-26), asshown in Scheme 10. The final compounds 1-20-1-25 were obtained in 55%,70%, 52%, 48%, 52% and 83% yield, respectively. Removal of ^(t)butylgroup from 1-25 furnished 1-26 in 94% yield.

(2-(((tert-Butyldimethylsilyl)oxy)methyl)-1′-tosyl-1′H-1,3′-bipyrrol-2′-yl)(2-methoxy-4-methylphenyl)methanol(3)

To a solution of 1-bromo-2-methoxy-4-methylbenzene (524 mg, 2.62 mmol)in anhydrous THF (5 mL) at −78° C. under N₂, n-BuLi (1.15 mL, 2.5 M inn-pentane, 2.88 mmol) was slowly added. After being stirred for 30 min,a solution of 2 (Cheng C, et al. Mar. Drugs. 2013, 11, 2927-2948) (600mg, 1.31 mmol) in anhydrous THF (1 mL) was added slowly via a syringe.The reaction was stirred for about 8 h and quenched by addition of asaturated aqueous NH₄Cl (15 mL) and extracted with EtOAc (10 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified quickly bycolumn chromatography (15% EtOAc/petroleum ether, R_(f)=0.3) to yield 3(unstable).

(2-(((tert-Butyldimethylsilyl)oxy)methyl)-1′-tosyl-1′H-1,3′-bipyrrol-2′-yl)(2-methoxy-4-methylphenyl)methanone(4)

To a solution of 3 in anhydrous DMSO (20 mL), IBX (618 mg, 2.20 mmol)was added at room temperature. The reaction was allowed to warm up to30° C. and stirred for about an additional 6 h. The reaction wasquenched with water (30 mL) and extracted with EtOAc (15 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by columnchromatography (15% EtOAc/petroleum ether, R_(f)=0.2) to yield 4 (550mg, 73% yield in two steps) as a pale brown solid: melting point30.7-33.0° C.; ¹H NMR (400 MHz, CDCl₃) δ 0.006 (s, 6H), 0.86 (s, 9H),2.38 (s, 3H), 2.40 (s, 3H), 3.75 (s, 3H), 4.67 (s, 2H), 6.20 (dd, J=4.0,2.4 Hz, 1H), 6.32 (d, J=3.2 Hz, 1H), 6.67 (dd, J=4.0, 1.6 Hz, 1H), 6.74(s, 1H), 6.75 (d, J=7.6 Hz, 1H), 7.06 (dd, J=2.4, 1.6 Hz, 1H), 7.18 (d,J=3.6 Hz, 1H), 7.24-7.28 (m, 3H), 7.84 (d, J=8.8 Hz, 2H) ppm; ¹³C NMR(CDCl₃, 100 MHz) δ −5.70, −5.70, 18.33, 21.46, 21.71, 25.80, 25.80,25.80, 53.45, 55.45, 108.90, 111.46, 112.05, 120.34, 121.16, 122.99,126.70, 126.86, 126.86, 127.20, 129.50, 129.64, 129.64, 129.81, 132.40,132.62, 136.66, 141.84, 144.60, 157.22, 184.12 ppm; HRMS ESI (M+H⁺)calculated for C₃₁H₃₉N₂O₅SSi 579.2349. found 579.2358; IR (KBr) 3434,3443, 2954, 2930, 2856, 1916, 1708, 1936, 1608, 1498, 1409, 1368, 1256,1179, 1035, 839, 772, 670, 602 cm⁻¹.

(2-(Hydroxymethyl)-1′-tosyl-1′H-1,3′-bipyrrol-2′-yl)(2-methoxy-4-methylphenyl)methanone(5)

To a solution of 4 (550 mg, 0.95 mmol) in anhydrous THF (10 mL), TBAF(745 mg, 2.85 mmol) was added at room temperature. The reaction wasallowed to stir for about an additional 5 h at room temperature. Thereaction was quenched with water (10 mL) and extracted with EtOAc (10mL×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated in vacuum. The residue was purifiedby column chromatography (20% EtOAc/petroleum ether, R_(f)=0.3) to yield5 (397 mg, 90% yield) as a brown-red solid: melting point 29.9-31.7° C.;¹H NMR (400 MHz, CDCl₃) δ 2.38 (s, 3H), 2.40 (s, 3H), 2.97 (t, J=6.8 Hz,1H), 3.74 (s, 3H), 4.55 (d, J=6.8 Hz, 2H), 6.23 (dd, J=4.0, 2.4 Hz, 1H),6.34 (d, J=3.6 Hz, 1H), 6.63 (dd, J=4.0, 1.6 Hz, 1H), 6.73 (s, 1H), 6.76(d, J=7.6 Hz, 1H), 7.00 (dd, J=2.4, 2.0 Hz, 1H), 7.22 (d, J=7.6 Hz, 1H),7.28 (d, J=3.6 Hz, 1H), 7.32 (d, J=8.0 Hz, 2H), 7.85 (d, J=8.4 Hz, 2H)ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 21.50, 21.70, 55.02, 55.48, 109.61,110.85, 112.06, 120.41, 121.03, 123.68, 126.61, 127.08, 127.08, 128.22,128.72, 129.27, 129.95, 129.95, 132.60, 132.82, 135.73, 141.93, 145.21,157.09, 185.16 ppm; HRMS ESI (M+Na⁺) calculated for C₂₅H₂₄N₂NaO₅S,487.1304. found 487.1297; IR (KBr) 3445, 3141, 2956, 2930, 1771, 1702,1631, 1609, 1498, 1461, 1410, 1366, 1177, 1138, 1034, 1014, 931, 720cm⁻¹.

2′-(2-Methoxy-4-methylbenzoyl)-1′-tosyl-1′H-1,3′-bipyrrole-2-carbaldehyde(6)

To a solution of 5 (413 mg, 0.89 mmol) in DMSO (20 mL), IBX (374 mg,1.33 mmol) was added at room temperature. The reaction was allowed towarm up to 50° C. and stirred for about 3 h. The reaction was quenchedwith water (30 mL) and extracted with EtOAc (15 mL×3). The combinedorganic layers were dried over anhydrous sodium sulfate, filtered andconcentrated in vacuum. The residue was purified by columnchromatography (15% EtOAc/petroleum ether, R_(f)=0.3) to yield 6 (370mg, 90% yield) as a white solid: melting point 116.4-118.0° C.; ¹H NMR(400 MHz, CDCl₃) δ 2.38 (s, 3H), 2.42 (s, 3H), 3.73 (s, 3H), 6.27 (s,1H), 6.45 (d, J=3.2 Hz, 1H), 6.68 (d, J=2.0 Hz, 1H), 6.72 (s, 1H), 6.76(d, J=7.6 Hz, 1H), 6.99 (s, 1H), 7.30 (d, J=8.0 Hz, 1H), 7.34 (d, J=8.0Hz, 2H), 7.68 (d, J=3.2 Hz, 1H), 7.89 (d, J=8.0 Hz, 2H), 9.62 (s, 1H)ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 21.53, 21.69, 55.41, 109.99, 111.43,112.02, 120.41, 123.04, 125.22, 125.99, 127.50, 127.94, 127.94, 129.80,129.83, 129.83, 131.70, 133.68, 134.72, 139.12, 142.26, 145.71, 157.32,176.91, 184.11 ppm; HRMS ESI (M+^(H)) calculated for C₂₅H₂₃N₂O₅S,463.1328. found 463.1336; IR (KBr) 3449, 3150, 3129, 2957, 2924, 2854,1690, 1671, 1606, 1565, 1408, 1357, 1263, 1170, 1073, 1009, 773 cm⁻¹.

(2-(Hydroxy(2-methoxy-4-methylphenyl)methyl)-1′-tosyl-1′H-1,3′-bipyrrol-2′-yl)(2-methoxy-4-methylphenyl)methanone(7)

To a solution of 1-bromo-2-methoxy-4-methylbenzene (378 mg, 1.89 mmol)in anhydrous THF (5 mL) at −78° C. under N₂, t-BuLi (1.46 mL, 1.3 M,1.89 mmol) was slowly added. After being stirred for 30 min, a solutionof 6 (350 mg, 0.76 mmol) in anhydrous THF (1 mL) was added slowly via asyringe. The reaction was stirred for about 8 h, quenched by addition ofa saturated aqueous NH₄Cl (15 mL), and extracted with EtOAc (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified quickly bycolumn chromatography (12% EtOAc/petroleum ether, R_(f)=0.3) to yield 7(unstable).

(1′-Tosyl-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-methoxy-4-methylphenyl)methanone)(8)

To a solution of 7 in anhydrous DMSO (20 mL), IBX (275 mg, 0.98 mmol)was added at room temperature. After being stirred for about 3 h, thereaction was quenched with water (30 mL) and extracted with EtOAc (15mL×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated in vacuum. The residue was purifiedby column chromatography (12% EtOAc/petroleum ether, R_(f)=0.2) to yield8 (240 mg, 54% yield two steps) as a pale brown solid: melting point71.0-72.7° C.; ¹H NMR (400 MHz, CDCl₃) δ 2.27 (s, 3H), 2.37 (s, 3H),3.43 (s, 3H), 3.65 (s, 3H), 3.75 (s, 3H), 5.85 (t, J=3.2 Hz, 1H), 6.29(dd, J=4.0, 1.6 Hz, 1H), 6.46-6.48 (m, 2H), 6.53 (d, J=8.0 Hz, 1H), 6.70(d, J=8.0 Hz, 1H), 6.73 (s, 2H), 6.96 (d, J=7.6 Hz, 1H), 7.28 (d, J=8.0Hz, 1H), 7.33 (d, J=8.0 Hz, 2H), 7.49 (d, J=3.6 Hz, 1H), 7.94 (d, J=8.0Hz, 2H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 21.50, 21.62, 21.79, 55.39,55.39, 108.52, 111.61, 111.99, 112.04, 119.88, 120.37, 122.95, 123.41,124.79, 126.25, 128.07, 128.07, 128.61, 129.41, 129.41, 129.41, 129.73,131.48, 132.17, 132.29, 135.78, 141.65, 144.24, 144.81, 157.25, 158.29,183.15, 184.47 ppm; HRMS ESI (M+H⁺) calculated for C₃₃H₃₁N₂O₆S. found583.1903. found 583.1890; IR (KBr) 3356, 3006, 2958, 2851, 1631, 1612,1463, 1408, 1262, 1157, 1088, 1033, 859, 746 cm⁻¹.

1′H-1,3′-bipyrrole-2,2′-diylbis((2-methoxy-4-methylphenyl)methanone) (9)

To a solution of 8 (210 mg, 0.36 mmol) in a mixture of MeOH/THF (1:1, 5mL), KOH (60 mg, 1.08 mmol) was added at room temperature. After beingstirred for 15 min, the reaction was adjusted to pH 7.0 with 0.5 N HCland extracted with EtOAc (10 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by column chromatography (33%EtOAc/petroleum ether, R_(f)=0.3) to yield 9 (151 mg, 98% yield) as alight white solid: melting point 171.1-172.3° C.; ¹H NMR (400 MHz,CDCl₃) δ 2.25 (s, 3H), 2.38 (s, 3H), 3.67 (s, 3H), 3.77 (s, 3H),5.80-5.82 (m, 1H), 6.30 (t, J=2.8 Hz, 1H), 6.35 (dd, J=4.0, 1.6 Hz, 1H),6.46-6.48 (m, 2H), 6.62 (t, J=2.8 Hz, 1H), 6.73-6.74 (m, 2H), 7.02 (t,J=2.8 Hz, 1H), 7.08 (dd, J=9.6, 7.6 Hz, 2H), 9.40 (br s, 1H) ppm; ¹³CNMR (CDCl₃, 100 MHz) δ 21.78, 21.81, 55.33, 55.58, 108.32, 110.60,111.35, 112.20, 119.99, 120.63, 122.76, 122.98, 125.23, 125.92, 126.77,129.01, 129.89, 131.13, 132.22, 132.34, 141.51, 141.55, 156.69, 157.43,183.43, 183.90 ppm; HRMS ESI (M+H⁺) calculated for C₂₆H₂₅N₂O₄ 429.1814.found 429.1811; IR (KBr) 3357, 3006, 2957, 2852, 1631, 1612, 1462, 1408,1264, 1127, 1033, 859, 747 cm⁻¹.

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((5-chloro-2-methoxy-4-methylphenyl)methanone)(10)

To a solution of 9 (10 mg, 0.02 mmol) in anhydrous MeCN (1 mL) at roomtemperature, NCS (18.7 mg, 0.14 mmol) was added slowly. After beingstirred for about 20 min at room temperature, the reaction was quenchedwith water (5 mL) and extracted with EtOAc (5 mL×3). The combinedorganic layers were dried over anhydrous sodium sulfate, filtered andconcentrated in vacuum. The residue was purified by columnchromatography (12% EtOAc/petroleum ether, R_(f)=0.2) to yield 10 (9 mg,65% yield) as a pale brown solid: melting point 78.3-80.0° C.; ¹H NMR(400 MHz, CDCl₃) δ 2.30 (s, 3H), 2.32 (s, 3H), 3.72 (s, 3H), 3.76 (s,3H), 6.44 (s, 1H), 6.67 (s, 1H), 6.80 (s, 1H), 7.06 (s, 1H), 7.20 (s,1H), 9.90-10.10 (br s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 20.54, 20.65,55.80, 55.89, 110.91, 111.82, 113.10, 114.04, 120.46, 120.80, 124.20,124.75, 124.87, 125.07, 125.31, 126.30, 128.27, 128.66, 129.86, 130.65,139.95, 140.16, 155.25, 156.06, 180.59, 180.86 ppm; HRMS ESI (M+H⁺)calculated for C₂₆H₁₉Cl₆N₂O₄ 632.9476. found 632.9492; IR (KBr) 3232,2955, 2918, 2849, 1736, 1644, 1604, 1462, 1428, 1401, 1172, 1039, 871,678 cm⁻¹.

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((5-chloro-2-hydroxy-4-methylphenyl)methanone)(1-2)

To a solution of 10 (47 mg, 0.07 mmol) in anhydrous CH₂Cl₂ (5 mL), asolution of BBr₃ (75 mg, 0.30 mmol) in anhydrous CH₂Cl₂ (1 mL) wasslowly added via a syringe under N₂ at −78° C. After being stirred for0.5 h, the reaction was quenched by addition of water (10 mL) andextracted with CH₂Cl₂ (10 mL×3). The combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated in vacuum. Theresidue was purified by column chromatography (12% EtOAc/petroleumether, R_(f)=0.2) to yield 10 (38 mg, 85% yield) as a pale brown solid:melting point 84.7-86.0° C.; ¹H NMR (400 MHz, CDCl₃) δ 2.32 (s, 3H),2.39 (s, 3H), 6.76 (s, 1H), 6.80 (s, 1H), 6.90 (s, 1H), 7.39 (s, 2H),9.89 (br s, 1H), 10.29 (s, 1H), 10.98 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100MHz) δ 20.72, 20.80, 113.28, 117.53, 117.77, 119.80, 119.82, 120.40,120.44, 120.74, 120.76, 123.34, 123.43, 124.38, 124.72, 128.60, 129.65,130.91, 145.51, 145.71, 159.52, 160.76, 184.34, 185.20 ppm; HRMS ESI(M+H⁺) calculated for C₂₄H₁₅C₁₆N₂O₄ 604.9163. found 604.9168; IR (KBr)3415, 3238, 2955, 2927, 2856, 1628, 1595, 1479, 1430, 1215, 1027, 871,690 cm⁻¹. HPLC purity, 95.6% (Flow rate: 1.0 mL/min; Column: AgilentZORBAX 300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature:25° C.; Mobile phase: MeOH:H₂O=75:25; t_(R)=9.0 min).

(4,4′,5,5′-Tetrachloro-1′-tosyl-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-methoxy-4-((trimethylsilyl)ethynyl)phenyl)methanone)(12)

Under N₂, a mixture of 11 (Cheng C, et al. Mar. Drugs. 2013, 11,2927-2948) (300 mg, 0.30 mmol), ethynyltrimethylsilane (176 mg, 1.80mmol), Pd(PPh₃)₄ (70 mg, 0.06 mmol) and Et₃N (30 mg, 0.30 mmol) wasdissolved in anhydrous DMF (5 mL). The reaction was allowed to stir forabout 16 h at room temperature. The reaction was quenched with water (15mL) and extracted with EtOAc (10 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by column chromatography (10%EtOAc/petroleum ether, R_(f)=0.3) to yield 12 (199 mg, 74% yield) as alight yellow solid: melting point 118.7-120.0° C.; ¹H NMR (400 MHz,acetone-d₆) δ 0.25 (s, 18H), 2.54 (s, 3H), 3.56 (s, 3H), 3.63 (s, 3H),6.52 (s, 1H), 6.69 (d, J=8.0 Hz, 1H), 6.93 (d, J=2.8 Hz, 2H), 7.04 (dd,J=8.0, 1.2 Hz, 1H), 7.13 (d, J=7.6 Hz, 1H), 7.49 (d, J=8.0 Hz, 1H), 7.60(d, J=8.0 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H) ppm; ¹³C NMR (CDCl₃, 100 MHz)δ −0.18, −0.18, −0.18, −0.14, −0.14, −0.14, 21.72, 56.11, 56.11, 96.70,97.96, 104.84, 105.05, 114.43, 114.66, 115.16, 116.20, 117.25, 118.20,120.38, 121.80, 123.55, 124.09, 124.78, 127.79, 127.98, 129.23, 129.23,129.80, 130.33, 131.02, 131.02, 131.10, 131.64, 134.42, 134.99, 147.58,158.07, 159.35, 181.79, 183.11 ppm; HRMS ESI (M+H⁺) calculated forC₄₁H₃₉Cl₄N₂O₆SSi₂ 883.0821. found 883.0812; IR (KBr) 3445, 2960, 2857,2159, 1654, 1600, 1556, 1456, 1400, 1268, 1250, 1192, 1034, 951, 851cm⁻¹.

(4,4′,5,5′-Tetrachloro-1′-tosyl-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-hydroxy-4-((trimethylsilyl)ethynyl)phenyl)methanone)(13)

To a solution of 12 (43 mg, 0.05 mmol) in anhydrous CH₂Cl₂ (5 mL), asolution of BBr₃ (61 mg, 0.24 mmol) in anhydrous CH₂Cl₂ (1 mL) wasslowly added via a syringe under N₂ at −78° C. After being stirred for30 min, the reaction was quenched by addition of water (10 mL) andextracted with CH₂Cl₂ (10 mL×3). The combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated in vacuum. Theresidue was purified by column chromatography (10% EtOAc/petroleumether, R_(f)=0.2) to yield 13 (22 mg, 53% yield) as a pale brown solid:melting point 97.3-99.7° C.; ¹H NMR (400 MHz, acetone-d₆) δ 0.27 (s,18H), 2.52 (s, 3H), 6.86 (s, 1H), 6.91-6.93 (m, 2H), 6.96 (d, J=8.8 Hz,1H), 7.44 (d, J=8.0 Hz, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.56 (d, J=8.0 Hz,3H), 7.90 (d, J=8.4 Hz, 2H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ −0.23,−0.23, −0.23, −0.23, −0.23, −0.23, 21.84, 99.67, 103.39, 103.72, 106.30,112.25, 113.64, 113.86, 120.07, 120.83, 121.20, 121.25, 121.35, 122.60,122.62, 122.80, 124.44, 128.25, 128.25, 128.26, 130.14, 130.14, 131.68,131.94, 131.98, 133.23, 133.80, 146.95, 146.95, 162.00, 162.00, 180.55,188.94 ppm; HRMS ESI (M+H⁺) calculated for C₃₉H₃₅Cl₄N₂O₆SSi₂ 855.0508.found 855.0502; IR (KBr) 2957, 2923, 2852, 2159, 1728, 1624, 1547, 1382,1343, 1245, 1191, 973, 850, 662 cm⁻¹.

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((4-ethynyl-2-hydroxyphenyl)methanone)(1-3)

To a solution of 13 (22 mg, 0.03 mmol) in a mixture of MeOH/THF (1:1, 3mL), KOH (7.2 mg, 0.13 mmol) was added at room temperature. After beingstirred for 15 min, the reaction was adjusted to pH 7.0 with 0.5 N HCland extracted with EtOAc (10 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by column chromatography (15%EtOAc/petroleum ether, R_(f)=0.2) to yield 1-3 (13 mg, 91% yield) as alight yellow solid: melting point 83.3-84.1° C.; ¹H NMR (400 MHz,acetone-d₆) δ 3.90 (s, 1H), 4.00 (s, 1H), 6.49 (s, 1H), 6.81 (d, J=8.0Hz, 1H), 7.02 (d, J=8.4 Hz, 1H), 7.05 (s, 1H), 7.54 (t, J=8.4 Hz, 3H),10.50 (s, 1H), 10.82 (s, 1H) 12.39 (br s, 1H) ppm; ¹³C NMR (CDCl₃, 100MHz) δ 78.81, 82.37, 82.99, 83.19, 109.91, 120.99, 121.00, 121.09,121.19, 121.81, 122.40, 122.75, 123.54, 124.80, 125.28, 129.32, 130.71,131.35, 134.03, 137.85, 141.50, 142.85, 161.00, 161.22, 185.62, 186.50ppm; HRMS ESI (M+H⁺) calculated for C₂₆H₁₃Cl₄N₂O₄ 556.9629. found556.9632; IR (KBr) 3405, 3295, 2969, 2929, 2108, 1701, 1624, 1594, 1551,1448, 1393, 1332, 1246, 1120, 965, 788, 675 cm⁻¹. HPLC purity, 99.1%(Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=70:30; t_(R)=5.7 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-hydroxy-4-vinylphenyl)methanone)(1-4)

Under 1 atm H₂, 1-3 (100 mg, 0.18 mmol) and Pd/BaSO₄ (5 mg) weredissolved in MeOH (3 mL). The reaction was allowed to cool to 10° C. andstirred for about 30 min. The suspension was filtered and the filtratewas washed with EtOAc (50 mL). The combined organic layers wereconcentrated in vacuum and the residue was purified by columnchromatography (20% EtOAc/petroleum ether, R_(f)=0.2) to yield 1-4 (61mg, 60% yield) as a yellow solid: melting point 76.4-77.7° C.; ¹H NMR(400 MHz, acetone-d₆) δ 5.41 (d, J=10.8 Hz, 1H), 5.49 (d, J=11.2 Hz,1H), 5.95 (d, J=17.6 Hz, 1H), 6.04 (d, J=17.6 Hz, 1H), 6.50 (s, 1H),6.95-6.79 (m, 2H), 6.81 (dd, J=8.0, 1.2 Hz, 1H), 6.98 (d, J=1.2 Hz, 1H),7.03 (s, 1H), 7.07 (d, J=8.4 Hz, 1H), 7.49 (d, J=7.2 Hz, 1H), 7.57 (d,J=8.4 Hz, 1H), 11.16 (s, 1H) ppm; ¹³C NMR (acetone-d₆, 100 MHz) δ 90.24,107.07, 109.66, 111.20, 115.52, 115.60, 117.12, 118.04, 118.27, 119.04,119.89, 128.70, 130.80, 131.83, 134.74, 136.58, 136.60, 137.88, 145.64,147.00, 152.18, 162.22, 171.91, 172.95, 185.92, 186.38 ppm; HRMS ESI(M+H⁺) calculated for C₂₆H₁₇Cl₄N₂O₄ 560.9942. found 560.9952; IR (KBr)3423, 3275, 2961, 2926, 1920, 1847, 1737, 1626, 1575, 1499, 1450, 1390,1353, 1216, 887, 797, 723 cm⁻¹. HPLC purity, 99.2% (Flow rate, 1.0mL/min; Column, Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6 mm; Wavelength:UV 254 nm; Temperature: 25° C.; Mobile phase: MeOH:H₂O=90:10; t_(R)=5.1min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((4-ethyl-2-hydroxyphenyl)methanone)(1-5)

Under 1 atm H₂, 1-4 (50 mg, 0.09 mmol) and Pd/BaSO₄ (5 mg) weredissolved in MeOH (3 mL). The reaction was allowed to stir for about 3 hat room temperature. The suspension was filtered and the filtrate waswashed with EtOAc (50 mL). The combined organic layers were concentratedin vacuum and the residue was purified by column chromatography (12%EtOAc/petroleum ether, R_(f)=0.2) to yield 1-5 (48 mg, 96% yield) as ayellow solid: melting point 90.3-92.0° C.; ¹H NMR (400 MHz, acetone-d₆)δ 1.17-1.24 (m, 6H), 2.59 (dd, J=15.2, 7.6 Hz, 2H), 2.65 (dd, J=15.2,7.6 Hz, 2H), 6.47 (s, 1H), 6.53 (dd, J=8.4, 2.8 Hz, 1H), 6.77-6.80 (m,3H), 7.35 (br s, 1H), 7.47 (d, J=8.0 Hz, 1H), 10.80 (br s, 1H), 11.24(s, 1H), 12.27 (br s, 1H) ppm; ¹³C NMR (acetone-d₆, 100 MHz) δ 15.01,15.01, 15.59, 15.59, 109.53, 117.00, 117.16, 118.24, 118.34, 119.10,120.31, 121.72, 124.79, 126.30, 128.10, 131.56, 131.56, 134.54, 154.47,154.47, 156.17, 156.17, 163.15, 163.41, 186.65, 188.42 ppm; HRMS ESI(M+H⁺) calculated for C₂₆H₂₁Cl₄N₂O₄ 565.0255. found 565.0261; IR (KBr)3420, 3251, 2967, 2932, 1628, 1590, 1500, 1450, 1393, 1258, 1124, 944,792, 531 cm⁻¹. HPLC purity, 97.5% (Flow rate: 1.0 mL/min; Column,Agilent ZORBAX 300SB-C8, 5 m, 150×4.6 mm; Wavelength: UV 254 nm;Temperature: 25° C.; Mobile phase: MeOH:H₂O=75:25; t_(R)=9.4 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(3-ethylacetate)thiophenyl)methanone)(1-9)

Under N₂, a mixture of 14 (Cheng C, et al. Mar. Drugs. 2013, 11,2927-2948) (50 mg, 0.06 mmol), ethyl 2-mercaptoacetate (33 mg, 0.24mmol), Pd₂(dba)₃ (2 mg, 0.003 mmol), Xantphos (4 mg, 0.006 mmol) andi-Pr₂NEt (31 mg, 0.24 mmol) was dissolved in 1,4-dioxane (5 mL). Thereaction was heated to reflux and stirred for about 10 h. The reactionwas quenched with water (10 mL) and extracted with EtOAc (15 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by flashcolumn chromatography (33% EtOAc/petroleum ether, R_(f)=0.3) to yield1-9 (28 mg, 61%) as a yellow solid: melting point 51.8-53.3° C.; ¹H NMR(400 MHz, CDCl₃) δ 1.24-1.29 (m, 6H), 3.71 (s, 2H), 3.76 (s, 2H),4.19-4.25 (m, 4H), 6.11 (s, 1H), 6.46 (d, J=8.4 Hz, 1H), 6.73 (d, J=8.0Hz, 1H), 6.80 (d, J=9.6 Hz, 2H), 7.20 (br s, 1H), 7.37 (br s, 1H), 10.51(br s, 1H), 10.93 (s, 1H), 11.58 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ14.01, 14.01, 33.92, 34.17, 62.05, 62.09, 108.68, 111.90, 113.64,114.13, 116.27, 116.27, 116.47, 116.80, 116.93, 118.74, 121.32, 122.17,124.17, 124.62, 130.62, 133.37, 147.17, 148.83, 161.82, 162.94, 168.61,168.70, 185.14, 186.83 ppm; HRMS ESI (M+^(H)) calculated forC₃₀H₂₅Cl₄N₂O₈S₂ 744.9806. found 744.9812; IR (KBr) 3671, 3368, 3080,2960, 2920, 1919, 1735, 1618, 1585, 1439, 1216, 1145, 1030, 745, 703cm⁻¹. HPLC purity, 96.8% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=90:10; t_(R)=20.0 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-benzylthio)phenyl)methanone)(1-10)

Under N₂, a mixture of 14 (Cheng C, et al. Mar. Drugs. 2013, 11,2927-2948) (50 mg, 0.06 mmol), phenylmethanethiol (32 mg, 0.24 mmol),Pd₂(dba)₃ (2 mg, 0.003 mmol), Xantphos (4 mg, 0.006 mmol) and i-Pr₂NEt(31 mg, 0.24 mmol) was dissolved in 1,4-dioxane (5 mL). The reaction washeated to reflux and stirred for about 4 h. The reaction was quenchedwith water (10 mL) and extracted with EtOAc (15 mL×3). The combinedorganic layers were dried over anhydrous sodium sulfate, filtered andconcentrated in vacuum. The residue was purified by flash columnchromatography (15% EtOAc/petroleum ether, R_(f)=0.3) to yield 1-10 (45mg, 96%) as a yellow solid: melting point 82.4-83.7° C.; ¹H NMR (400MHz, CDCl₃) δ 4.17 (s, 2H), 4.22 (s, 2H), 6.11 (s, 1H), 6.47 (d, J=8.4Hz, 1H), 6.61 (br, s, 1H), 6.74 (s, 1H), 6.80 (s, 1H), 7.27-7.41 (m,12H), 9.90 (br s, 1H), 10.95 (s, 1H), 11.63 (s, 1H) ppm; ¹³C NMR (CDCl₃,100 MHz) δ 36.24, 36.43, 108.77, 111.98, 113.35, 113.85, 115.84, 116.15,116.26, 116.79, 117.01, 121.27, 122.15, 124.21, 124.72, 127.65, 127.65,127.68, 127.68, 128.68, 128.68, 128.68, 128.78, 128.78, 128.78, 128.78,130.46, 133.14, 135.34, 135.49, 149.49, 151.08, 161.84, 163.04, 185.08,186.71 ppm; HRMS ESI (M+H⁺) calculated for C₃₆H₂₅Cl₄N₂O₄S₂ 753.0010.found 753.0005; IR (KBr) 3410, 3236, 3061, 3028, 2923, 1616, 1581, 1483,1448, 1391, 1327, 1223, 1073, 928, 780 cm⁻¹. HPLC purity, 96.9% (Flowrate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6 mm;Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase: MeOH:H₂O=95:5;t_(R)=6.6 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(4-methoxybenzylthio)phenyl)methanone)(1-11)

Under N₂, a mixture of 14 (Cheng C, et al. Mar. Drugs. 2013, 11,2927-2948) (20 mg, 0.02 mmol), (4-methoxyphenyl) methanethiol (15 mg,0.10 mmol), Pd₂(dba)₃ (0.8 mg, 0.001 mmol), Xantphos (1.6 mg, 0.002mmol) and i-Pr₂NEt (12 mg, 0.10 mmol) was dissolved in 1,4-dioxane (3mL). The reaction was heated to reflux and stirred for about 4 h. Thereaction was quenched with water (10 mL) and extracted with EtOAc (10mL×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated in vacuum. The residue was purifiedby flash column chromatography (20% EtOAc/petroleum ether, R_(f)=0.3) toyield 1-11 (17 mg, 85%) as a yellow solid: melting point 85.4-86.7° C.;¹H NMR (400 MHz, CDCl₃) δ 3.78 (s, 3H), 3.79 (s, 3H), 4.13 (s, 2H), 4.18(s, 2H), 6.11 (s, 1H), 6.46 (d, J=8.4 Hz, 1H), 6.62 (s, 1H), 6.74 (s,1H), 6.80 (s, 1H), 6.86 (dd, J=8.4, 3.2 Hz, 5H), 7.31 (t, J=8.4 Hz, 5H),9.71 (br s, 1H), 10.99 (s, 1H), 11.64 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100MHz) δ 35.72, 35.90, 53.66, 55.25, 108.83, 111.94, 113.30, 113.83,114.18, 114.18, 114.18, 114.18, 114.18, 115.78, 116.11, 116.24, 116.72,117.01, 121.23, 121.96, 124.08, 124.77, 127.11, 127.29, 129.85, 129.85,129.96, 129.96, 130.40, 133.12, 149.68, 151.31, 159.01, 159.06, 161.90,163.05, 185.02, 186.71 ppm; HRMS ESI (M+H⁺) calculated forC₃₈H₂₉Cl₄N₂O₆S₂ 813.0221. found 813.0228; IR (KBr) 3421, 3253, 2929,2836, 1702, 1615, 1511, 1447, 1393, 1329, 1248, 1177, 1075, 1034, 943cm⁻¹. HPLC purity, 96.5% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=95:5; t_(R)=6.4 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(3-ethylacetate)sulfonylphenyl)methanone)(1-12)

To a solution of 1-9 (22 mg, 0.03 mmol) in CH₂Cl₂ (2 mL), a solution ofm-CPBA (31 mg, 0.18 mmol) in CH₂Cl₂ (1 mL) was added at roomtemperature. After being stirred for about 20 h, the reaction wasquenched by addition water (10 mL) and extracted with CH₂Cl₂ (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by flashcolumn chromatography (20% acetone, 27% EtOAc, 53% petroleum ether,R_(f)=0.2) to yield 1-12 (16.7 mg, 70%) as a yellow solid: melting point90.0-91.7° C.; ¹H NMR (400 MHz, acetone-d₆) δ 1.16-1.18 (m, 6H),4.09-4.14 (m, 4H), 4.33 (s, 2H), 4.42 (s, 2H), 6.48 (s, 1H), 7.32 (d,J=8.0 Hz, 1H), 7.45-7.52 (m, 3H), 7.61 (d, J=8.0 Hz, 1H), 7.23 (d, J=8.0Hz, 1H), 10.48 (br s, 2H), 12.33 (br s, 1H) ppm; ¹³C NMR (acetone-d₆,100 MHz) δ 14.14, 14.14, 60.98, 61.02, 62.58, 62.58, 110.42, 112.21,117.11, 117.38, 118.94, 119.29, 119.65, 122.89, 124.02, 126.06, 127.21,128.65, 128.75, 129.08, 131.08, 132.87, 144.04, 144.64, 157.98, 158.59,163.00, 163.05, 183.41, 184.48 ppm; HRMS ESI (M+H⁺) calculated forC₃₀H₂₅Cl₄N₂O₁₂S₂ 808.9603. found 808.9599; IR (KBr) 2918, 2850, 2490,1741, 1637, 1589, 1441, 1407, 1330, 1269, 1215, 1151, 1029, 751, 703,637 cm⁻¹. HPLC purity, 98.1% (Flow rate: 1.0 mL/min; Column: Waters C18,5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobilephase: MeOH:H₂O=70:30; t_(R)=30.4 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-benzylsulfonyl)phenyl)methanone)(1-13)

To a solution of 1-10 (30 mg, 0.04 mmol) in CH₂Cl₂ (2 mL), a solution ofm-CPBA (42 mg, 0.24 mmol) in CH₂Cl₂ (1 mL) was added at roomtemperature. After being stirred for about 5 h, the reaction wasquenched by addition water (10 mL) and extracted with CH₂Cl₂ (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by flashcolumn chromatography (20% acetone, 27% EtOAc, 53% petroleum ether,R_(f)=0.2) to yield 1-13 (24 mg, 75%) as a yellow solid: melting point130.9-132.7° C.; ¹H NMR (400 MHz, CDCl₃) δ 4.26 (s, 2H), 4.34 (s, 2H),6.12 (s, 1H), 6.99 (d, J=8.4 Hz, 1H), 7.05 (d, J=8.4 Hz, 1H), 7.14-7.17(m, 4H), 7.29-7.37 (m, 8H), 7.58 (d, J=8.4 Hz, 1H), 7.68 (d, J=8.0 Hz,1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 62.17, 62.35, 110.30, 110.34,112.10, 117.23, 117.51, 118.83, 119.18, 119.80, 124.03, 126.02, 127.12,127.83, 128.40, 128.75, 129.07, 129.15, 129.15, 129.19, 129.19, 129.34,129.40, 129.45, 131.25, 131.88, 131.88, 131.88, 131.98, 131.98, 132.82,144.07, 144.21, 158.24, 183.74, 184.70 ppm; HRMS ESI (M+H⁺) calculatedfor C₃₆H₂₅Cl₄N₂O₈S₂ 816.9806. found 816.9800; IR (KBr) 3078, 3030, 2959,2920, 2851, 2583, 1730, 1636, 1591, 1142, 1407, 1319, 1149, 1125, 879,750, 701, 628 cm⁻¹. HPLC purity, 97.1% (Flow rate: 1.0 mL/min; Column:Agilent ZORBAX 300SB-C8, 5 m, 150×4.6 mm; Wavelength: UV 254 nm;Temperature: 25° C.; Mobile phase: MeOH:H₂O=60:40; t_(R)=11.9 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(4-methoxybenzylsulfonyl)phenyl)methanone)(1-14)

To a solution of 1-11 (60 mg, 0.07 mmol) in CH₂Cl₂ (5 mL), a solution ofm-CPBA (128 mg, 0.74 mmol) in CH₂Cl₂ (2 mL) was added at roomtemperature. After being stirred for about 20 h, the reaction wasquenched by adding water (10 mL) and extracted with CH₂Cl₂ (10 mL×3).The combined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by flashcolumn chromatography (20% acetone, 27% EtOAc, 53% petroleum ether,R_(f)=0.2) to yield 1-14 (42 mg, 65%) as a yellow solid: melting point104.3-106.3° C.; ¹H NMR (400 MHz, CDCl₃) δ 3.77 (s, 6H), 4.20 (s, 2H),4.31 (s, 2H), 6.17 (s, 1H), 6.80 (t, J=7.2 Hz, 4H), 6.97 (d, J=8.4 Hz,1H), 7.05 (d, J=8.4 Hz, 5H), 7.16 (s, 1H), 7.36 (s, 1H), 7.62 (d, J=8.4Hz, 1H), 7.67 (d, J=8.0 Hz, 1H), 10.44 (br s, 1H), 11.00 (br s, 1H) ppm;¹³C NMR (CDCl₃, 100 MHz) δ 55.26, 55.26, 61.84, 61.84, 109.45, 112.63,114.18, 114.18, 114.18, 114.22, 114.22, 117.59, 118.33, 118.57, 118.57,118.82, 118.93, 122.16, 122.80, 123.45, 124.62, 125.25, 126.44, 131.09,132.02, 132.02, 132.02, 132.09, 132.09, 133.78, 143.91, 144.84, 160.15,160.22, 160.63, 161.77, 184.85, 187.06 ppm; HRMS ESI (M+Na⁺) calculatedfor C₃₈H₂₈Cl₄N₂NaO₁₀S₂ 898.9837. found 898.9838; IR (KBr) 2961, 2920,2850, 1730, 1632, 1592, 1512, 1444, 1257, 1148, 1099, 1030, 798 cm⁻¹.HPLC purity, 95.1% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=65:35; t_(R)=6.0 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(p-aceticacid)thiophenyl)methanone) (1-15)

To a solution of 1-9 (20 mg, 0.03 mmol) in a mixture of H₂O/THF (1:2, 3mL), LiOH (15 mg, 0.35 mmol) was added at room temperature. The reactionwas allowed to warm up to 50° C. and stirred for about 10 h. Thereaction was adjusted to pH 5.0 with 0.5 N HCl and extracted with EtOAc(5 mL×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated in vacuum. The residue was purifiedby reverse-phase column chromatography (C18 reverse silica gel, 7% AcOH,22% H₂O, 71% MeOH, R_(f)=0.2) to yield 1-15 (15.7 mg, 85%) as a brownsolid: melting point 137.9-139.7° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 3.46(s, 2H), 3.65 (s, 2H), 6.03 (s, 1H), 6.53-6.60 (m, 3H), 6.64 (s, 1H),7.38 (d, J=7.6 Hz, 1H), 7.85 (d, J=8.0 Hz, 1H) ppm; ¹³C NMR (DMSO-d₆,100 MHz) δ 36.10, 36.80, 108.30, 109.39, 112.71, 112.94, 115.56, 116.02,116.84, 118.22, 120.95, 122.60, 123.09, 124.39, 128.56, 131.13, 132.65,133.61, 144.10, 149.87, 159.26, 160.70, 171.50, 172.90, 181.57, 186.55ppm; HRMS ESI (M+Na⁺) calculated for C₂₆H₁₆Cl₄N₂NaO₈S₂ 710.9000. found710.9009; IR (KBr) 3392, 2955, 2918, 2849, 1592, 1382, 1223, 1023, 671cm⁻¹. HPLC purity, 98.7% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX300SB-C8, 5 m, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=60:40; t_(R)=8.0 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis(((2-hydroxy-4-(p-aceticacid)sulfonylphenyl)methanone) (1-16)

To a solution of 1-12 (70 mg, 0.09 mmol) in a mixture of H₂O/THF (1:2, 5mL), LiOH (49 mg, 1.13 mmol) was added at room temperature. The reactionwas allowed to warm up to 50° C. and stir for about 3 h. The reactionwas adjusted to pH 5.0 with 0.5 N HCl and extracted with EtOAc (10mL×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated in vacuum. The residue was purifiedby reverse column chromatography (C18 reverse silica gel, 4% AcOH, 38%H₂O, 58% MeOH, R_(f)=0.2) to yield 1-16 (62 mg, 95%) as a yellow solid:melting point 143.2-144.7° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 4.12 (s, 2H),4.18 (s, 2H), 6.22 (s, 1H), 7.15 (d, J=8.0 Hz, 1H), 7.27-7.29 (m, 4H),7.36-7.40 (m, 1H) ppm; ¹³C NMR (acetone-d₆, 100 MHz) δ 52.09, 52.09,100.10, 100.18, 100.98, 105.79, 107.20, 108.26, 108.65, 112.73, 113.40,115.44, 118.90, 118.91, 120.25, 120.40, 120.53, 121.64, 133.09, 146.18,146.89, 154.58, 163.09, 163.09, 171.58, 172.34 ppm; HRMS ESI (M+H⁺)calculated for C₂₆H₁₇Cl₄N₂O₁₂S₂ 752.8977. found 752.8981; IR (KBr) 3395,2957, 2923, 1628, 1445, 1407, 1313, 1147, 1026, 1000, 906, 825, 701cm⁻¹. HPLC purity, 95.2% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=60:40; t_(R)=4.0 min).

(1′-Tosyl-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-methoxy-4-((trimethylsilyl)ethynyl)phenyl)methanone)(16)

Under N₂, a mixture of 15 (Cheng C, et al. Mar. Drugs. 2013, 11,2927-2948) (50 mg, 0.06 mmol), ethynyltrimethylsilane (34 mg, 0.35mmol), Pd(PPh₃)₄ (15 mg, 0.01 mmol) and Et₃N (18 mg, 0.18 mmol) wasdissolved in anhydrous DMF (5 mL). The reaction was heated to 60° C. andstirred for 10 h. The reaction was quenched with water (10 mL) andextracted with EtOAc (15 mL×3). The combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated in vacuum. Theresidue was purified by column chromatography (10% EtOAc/petroleum,R_(f)=0.3) to yield 16 (40 mg, 92% yield) as a brown solid: meltingpoint 105.3-106.7° C.; ¹H NMR (400 MHz, CDCl₃) δ 0.07 (s, 6H), 0.26 (s,6H), 0.27 (s, 6H), 2.44 (s, 3H), 3.65 (s, 3H), 3.77 (s, 3H), 5.90 (t,J=2.8 Hz, 1H), 6.29 (dd, J=4.0, 1.2 Hz, 1H), 6.44 (d, J=3.2 Hz, 1H),6.71 (br s, 2H), 6.79 (d, J=7.6 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H), 6.99(s, 1H), 7.04 (d, J=7.6 Hz, 1H), 7.21 (d, J=7.6 Hz, 1H), 7.35 (d, J=8.0Hz, 2H), 7.56 (d, J=3.2 Hz, 1H), 7.94 (d, J=8.4 Hz, 2H) ppm; ¹³C NMR(CDCl₃, 100 MHz) δ −0.13, −0.13, −0.13, −0.13, −0.13, −0.13, −21.71,55.67, 55.67, 95.75, 96.80, 104.23, 104.31, 109.13, 111.89, 114.16,114.39, 123.46, 123.55, 123.55, 124.47, 125.81, 127.61, 127.65, 128.31,128.31, 128.52, 129.06, 129.63, 129.63, 129.90, 130.86, 131.92, 132.76,133.02, 135.74, 145.16, 156.89, 157.58, 182.54, 184.05 ppm; HRMS ESI(M+H⁺) calculated for C₄₁H₄₃N₂O₆SSi₂ 747.2380. found 747.2382; IR (KBr)3443, 3145, 2959, 2857, 2158, 1649, 1600, 1556, 1405, 1377, 1272, 1253,1175, 1135, 1034, 952, 852, 667 cm⁻¹.

1′H-1,3′-Bipyrrole-2,2′-diylbis((4-ethynyl-2-methoxyphenyl)methanone)(17)

To a solution of 16 (300 mg, 0.40 mmol) in a mixture of MeOH/THF (1:1,10 mL), KOH (113 mg, 2.0 mmol) was added at room temperature. Afterbeing stirred for 2 h, the reaction was adjusted to pH 7.0 with 0.5 NHCl and extracted with EtOAc (10 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by column chromatography (33%EtOAc/petroleum ether, R_(f)=0.3) to yield 17 (171 mg, 95% yield) as abrown solid: melting point 178.7-180.0° C.; ¹H NMR (400 MHz, CDCl₃) δ3.10 (s, 1H), 3.15 (s, 1H), 3.69 (s, 3H), 3.79 (s, 3H), 5.88 (dd, J=4.0,2.4 Hz, 1H), 6.31 (t, J=2.4 Hz, 1H), 6.36 (dd, J=4.0, 1.6 Hz, 1H), 6.64(t, J=2.0 Hz, 1H), 6.78 (s, 1H), 6.79 (d, J=7.6 Hz, 1H), 7.04 (s, 1H),7.08 (t, J=2.8 Hz, 1H), 7.10-7.12 (m, 3H), 9.43 (br s, 1H) ppm; ¹³C NMR(DMSO-d₆+acetone-d₆, 100 MHz) δ 55.39, 55.65, 80.75, 80.98, 83.21,83.38, 108.79, 109.96, 114.08, 114.94, 122.74, 123.51, 123.68, 123.76,124.20, 124.45, 126.25, 128.65, 129.17, 129.75, 130.70, 131.13, 132.10,133.06, 156.16, 156.69, 181.92, 182.70 ppm; HRMS ESI (M+H⁺) calculatedfor C₂₈H₂₁N₂O₄ 449.1501. found 449.1494; IR (KBr) 3339, 3281, 3259,3130, 2942, 2855, 1645, 1612, 1559, 1494, 1409, 1263, 1121, 938, 749cm⁻¹.

Diethyl2,2′-(4,4′-((1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(3-methoxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(18)

Under N₂, a mixture of 17 (50 mg, 0.11 mmol), ethyl 2-azidoacetate (58mg, 0.44 mmol), and CuCl (10 mg, 0.11 mmol) was dissolved in THF (5 mL).The reaction was allowed to warm up to reflux and stirred for about 10h. The suspension was filtered and the filtrate was concentrated invacuum. The residue was purified by column chromatography (33%EtOAc/petroleum ether, R_(f)=0.3) to yield 18 (61 mg, 78% yield) as alight yellow solid: melting point 136.3-137.7° C.; ¹H NMR (400 MHz,CDCl₃) δ 1.31-1.41 (m, 6H), 3.78 (s, 3H), 3.87 (s, 3H), 4.27-4.33 (m,4H), 5.22 (s, 4H), 5.87 (dd, J=4.0, 2.8 Hz, 1H), 6.33 (t, J=2.4 Hz, 1H),6.36 (dd, J=4.0, 1.6, 1H), 6.71 (t, J=2.4 Hz, 1H), 7.01 (d, J=7.6 Hz,1H), 7.07 (t, J=3.2 Hz, 1H), 7.17 (d, J=7.6 Hz, 1H), 7.22-7.24 (m, 2H),7.36 (s, 1H), 7.55 (s, 1H), 7.89 (s, 1H), 8.00 (s, 1H), 9.45 (br s, 1H)ppm; ¹³C NMR (DMSO-d₆+CD₃OD, 100 MHz) δ 14.51, 14.59, 51.80, 51.80,56.04, 56.28, 63.15, 63.15, 108.73, 109.47, 110.08, 111.05, 117.76,118.22, 124.40, 124.50, 124.55, 124.59, 124.59, 127.38, 129.49, 130.13,130.54, 131.28, 132.62, 133.40, 134.38, 134.48, 134.55, 147.93, 158.25,158.82, 168.38, 168.43, 184.34, 184.86 ppm; HRMS ESI (M+H⁺) calculatedfor C₃₆H₃₅N₈O₈ 707.2578. found 707.2588; IR (KBr) 3420, 3265, 3139,2986, 2942, 2852, 1745, 1634, 1614, 1562, 1412, 1247, 1226, 1132, 1028,932, 783 cm⁻¹.

Diethyl2,2′-(4,4′-((4,4′,5,5′-tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(2-chloro-5-methoxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(19)

To a solution of 18 (40 mg, 0.06 mmol) in AcOH (3 mL) at roomtemperature, NCS (54 mg, 0.40 mmol) was added slowly. The reaction wasallowed to stir for about 8 h at room temperature. The reaction wasquenched with water (15 mL) and extracted with EtOAc (10 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by columnchromatography (25% EtOAc/petroleum ether, R_(f)=0.2) to yield 19 (5 mg,10% yield) as a brown solid: melting point 118.7-120.3° C.; ¹H NMR (400MHz, CDCl₃) δ 1.28-1.36 (m, 6H), 3.84 (s, 3H), 3.87 (s, 3H), 4.25-4.33(m, 4H), 5.23 (s, 2H), 5.27 (s, 2H), 6.49 (s, 1H), 7.23 (s, 1H), 7.36(s, 1H), 7.79 (s, 1H), 7.90 (s, 1H), 8.36 (s, 1H), 8.45 (s, 1H), 10.86(br s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 29.59, 29.63, 50.94, 51.00,56.11, 56.14, 62.52, 62.58, 111.11, 111.44, 112.20, 112.31, 120.78,121.14, 121.59, 121.82, 124.20, 125.09, 125.19, 125.24, 125.28, 126.76,128.03, 129.63, 130.51, 131.09, 131.71, 131.93, 143.50, 143.66, 155.59,156.17, 166.04, 166.10, 180.07, 180.49 ppm; HRMS ESI (M+H⁺) calculatedfor C₃₆H₂₉Cl₆N₈O, 911.0240. found 911.0265; IR (KBr) 3419, 3162, 2925,2852, 1750, 1645, 1607, 1463, 1396, 1254, 1218, 1022, 915 cm⁻¹.

Diethyl2,2′-(4,4′-((4,4′,5,5′-tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(2-chloro-5-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(1-17)

To a solution of 19 (40 mg, 0.04 mmol) in anhydrous CH₂Cl₂ (5 mL), asolution of BBr₃ (44 mg, 0.16 mmol) in anhydrous CH₂Cl₂ (1 mL) wasslowly added via a syringe under N₂ at −78° C. After being stirred for 2h, the reaction was quenched by addition of water (10 mL) and extractedwith CH₂Cl₂ (10 mL×3). The combined organic layers were dried overanhydrous sodium sulfate, filtered and concentrated in vacuum. Theresidue was purified by column chromatography (30% EtOAc/petroleumether, R_(f)=0.2) to yield 1-17 (19 mg, 50% yield) as a brown solid:melting point 157.3-169.7° C.; ¹H NMR (400 MHz, CDCl₃) δ 1.29-1.35 (m,6H), 4.25-4.34 (m, 4H), 5.21 (s, 2H), 5.25 (s, 2H), 6.81 (s, 1H), 7.65(br s, 2H), 7.92 (s, 1H), 7.98 (s, 1H), 8.37 (s, 1H), 8.46 (s, 1H),10.91 (br s, 1H) ppm; ¹³C NMR (acetone-d₆, 100 MHz) δ 14.31, 14.31,51.39, 51.49, 62.40, 62.46, 109.10, 111.18, 118.09, 118.25, 118.87,118.95, 119.75, 120.46, 120.93, 121.53, 123.79, 126.17, 126.71, 126.92,127.51, 131.06, 134.03, 134.39, 134.61, 136.25, 143.15, 143.78, 158.99,160.48, 167.59, 167.67, 181.63, 186.11 ppm; HRMS ESI (M+H⁺) calculatedfor C₃₄H₂₅Cl₆N₈O₈ 882.9927. found 882.9933; IR (KBr) 3446, 2955, 2923,2850, 1749, 1627, 1458, 1377, 1218, 1020, 919, 775 cm⁻¹. HPLC purity,95.2% (Flow rate: 1.0 mL/min; Column: Phenomenex C6-phenyl, 5 μm,150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=80:20; t_(R)=10.8 min).

(1′-Tosyl-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene)bis(trifluoromethanesulfonate)(20)

To a solution of 15 (Cheng C, et al. Mar. Drugs. 2013, 11, 2927-2948)(2.5 g, 2.94 mmol) in anhydrous CH₂Cl₂ (100 mL), a solution of BBr₃(3.68 g, 14.70 mmol) in anhydrous CH₂Cl₂ (5 mL) was slowly added via asyringe under N₂ at −78° C. After being stirred for 0.5 h, the reactionwas quenched by addition of water (100 mL) and extracted with CH₂Cl₂ (50mL×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated in vacuum. The residue was purifiedby column chromatography (12% EtOAc/petroleum ether, R_(f)=0.3) to yield20 (2.17 g, 90% yield) as a yellow oil: ¹H NMR (400 MHz, CDCl₃) δ 2.48(s, 3H), 6.15 (t, J=2.8 Hz, 1H), 6.40 (d, J=3.6 Hz, 1H), 6.45 (dd,J=8.8, 2.4 Hz, 1H), 6.70 (d, J=3.6 Hz, 1H), 6.75 (d, J=2.4 Hz, 1H), 6.78(dd, J=8.8, 2.4 Hz, 1H), 6.84 (s, 1H), 6.91 (d, J=2.0 Hz, 1H), 7.39 (d,J=8.0 Hz, 2H), 7.56 (s, 1H), 7.57 (d, J=5.2 Hz, 1H), 7.68 (d, J=8.8 Hz,1H), 7.89 (d, J=8.4 Hz, 2H), 11.48 (s, 1H), 11.83 (s, 1H) ppm; ¹³C NMR(CDCl₃, 100 MHz) δ 21.70, 110.72, 110.72, 111.03, 111.09, 111.72,112.06, 119.24, 119.59, 123.57, 123.57, 125.45, 128.16, 128.16, 128.40,129.93, 129.93, 130.00, 131.77, 131.85, 133.88, 133.88, 134.48, 135.00,146.15, 153.58, 153.98, 163.57, 164.06, 186.46, 189.44 ppm. HRMS ESI(M+H⁺) calculated for C₃₁H₂₁F₆N₂O₁₂S₃ 823.0161. found 823.0173. IR (KBr)3159, 2943, 1685, 1671, 1550, 1454, 1368, 1272, 1137, 1027, 899 cm⁻¹.

(1′-Tosyl-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-hydroxy-4-((trimethylsilyl)ethynyl)phenyl)methanone)(21)

Under N₂, a mixture of 20 (100 mg, 0.12 mmol), ethynyltrimethylsilane(72 mg, 0.73 mmol), Pd(PPh₃)₄ (30 mg, 0.02 mmol) and Et₃N (37 mg, 0.36mmol) was dissolved in anhydrous DMF (5 mL). The reaction was allowed towarm up 15 to 70° C. and stirred for about 10 h. The reaction wasquenched with water (10 mL) and extracted with EtOAc (10 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by columnchromatography (10% EtOAc/petroleum ether, R_(f)=0.3) to yield 21 (85mg, 98% yield) as a pale yellow oil: ¹H NMR (400 MHz, CDCl₃) δ 0.23 (s,9H), 0.26 (s, 9H), 2.45 (s, 3H), 6.09 (t, J=2.8 Hz, 1H), 6.39 (d, J=3.2Hz, 1H), 6.56 (d, J=8.0 Hz, 1H), 6.65 (d, J=3.2 Hz, 1H), 6.77 (s, 1H),6.90 (s, 1H), 6.92 (d, J=8.8 Hz, 1H), 7.06 (s, 1H), 7.34-7.38 (m, 3H),7.48 (d, J=3.6 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.86 (d, J=8.0 Hz, 2H),11.34 (s, 1H), 11.55 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ −0.26,−0.26, −0.26, −0.21, −0.21, −0.21, 21.76, 98.47, 99.46, 103.56, 103.78,110.47, 110.95, 119.48, 119.66, 120.61, 121.14, 122.07, 122.55, 123.15,123.92, 124.19, 128.14, 128.14, 129.86, 129.86, 130.08, 130.35, 131.10,131.40, 131.48, 131.98, 132.49, 135.18, 145.91, 161.91, 162.05, 187.11,190.31 ppm; HRMS ESI (M+H⁺) calculated for C₃₉H₃₉N₂O₆SSi₂ 719.2067.found 719.2062. IR (KBr) 3159, 2997, 2973, 1795, 1681, 1580, 1417, 1272,1167, 1097, 878 cm⁻¹.

1′H-1,3′-Bipyrrole-2,2′-diylbis((4-ethynyl-2-hydroxyphenyl)methanone)(22)

To a solution of 21 (85 mg, 0.12 mmol) in a mixture of MeOH/THF (1:1, 5mL), KOH (33 mg, 0.59 mmol) was added at room temperature. After beingstirred for 1.5 h, the reaction was adjusted to pH 7.0 with 0.5 N HCland extracted with EtOAc (10 mL×3). The combined organic layers weredried over anhydrous sodium sulfate, filtered and concentrated invacuum. The residue was purified by column chromatography (33%EtOAc/petroleum ether, R_(f)=0.3) to yield 22 (47 mg, 95% yield) as ayellow oil: ¹H NMR (400 MHz, CDCl₃) δ 3.20 (s, 1H), 3.24 (s, 1H), 6.23(t, J=3.2 Hz, 1H), 6.36 (s, 1H), 6.55 (d, J=8.0 Hz, 1H), 6.72 (d, J=3.2Hz, 1H), 6.90 (d, J=6.8 Hz, 1H), 6.93 (d, J=4.0 Hz, 2H), 7.07 (s, 1H),7.14 (t, J=2.8 Hz, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.32 (d, J=8.0 Hz, 1H),9.48 (br s, 1H), 10.97 (s, 1H), 11.48 (s, 1H) ppm; ¹³C NMR (acetone-d₆,100 MHz) δ 82.18, 82.40, 83.10, 109.73, 109.79, 110.96, 120.91, 121.00,121.06, 121.15, 121.24, 122.49, 122.57, 123.83, 124.92, 125.09, 128.81,129.37, 131.01, 131.37, 133.14, 161.25, 162.14, 162.42, 187.26, 187.82ppm; HRMS ESI (M+Na⁺) calculated for C₂₆H₁₆N₂NaO₄ 443.1008. found443.1003. IR (KBr) 3435, 3239, 2980, 1785, 1691, 1590, 1424, 1127, 1017,886 cm⁻¹.

Di-tert-butyl2,2′-(4,4′-((1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(23)

Under N₂, a mixture of 22 (200 mg, 0.48 mmol), tert-butyl 2-azidoacetate(300 mg, 1.90 mmol), and CuCl (47 mg, 0.48 mmol) was dissolved in THF (5mL). The reaction was allowed to warm up to reflux and stirred for about8 h. The suspension was filtered and the filtrate was concentrated invacuum. The residue was purified by column chromatography (40%EtOAc/petroleum ether, R_(f)=0.3) to yield 23 (280 mg, 80% yield) as ayellow solid: melting point 96.0-97.0° C.; ¹H NMR (400 MHz, acetone-d₆)δ 1.48 (s, 9H), 1.49 (s, 9H), 5.31 (s, 2H), 5.33 (s, 2H), 6.28 (dd,J=3.6, 2.8 Hz, 1H), 6.44 (t, J=2.4 Hz, 1H), 6.77 (dd, J=4.0, 1.6 Hz,1H), 7.10 (dd, J=8.4, 1.6 Hz, 1H), 7.26-7.32 (m, 4H), 7.44 (d, J=1.6 Hz,1H), 7.48-7.51 (m, 2H), 8.49 (s, 1H), 8.52 (s, 1H), 11.29 (br s, 1H),11.36 (s, 1H), 11.83 (s, 1H) ppm; ¹³C NMR (acetone-d₆, 100 MHz) δ 28.03,28.03, 28.03, 28.03, 28.03, 28.03, 52.10, 52.10, 83.40, 83.40, 109.66,110.87, 114.19, 114.38, 116.28, 116.29, 119.66, 119.89, 123.34, 123.35,124.09, 124.44, 124.48, 124.67, 130.89, 131.29, 132.42, 132.74, 134.03,138.29, 138.58, 146.62, 162.93, 163.74, 166.68, 166.71, 188.03, 188.48ppm; HRMS ESI (M+H⁺) calculated for C₃₈H₃₉N₈O₈ 735.2891. found 735.2894;IR (KBr) 3405, 3139, 2977, 2933, 1745, 1631, 1590, 1414, 1368, 1242,1157, 1047, 898, 793 cm⁻¹.

Di-tert-butyl2,2′-(4,4′-((4,4′,5,5′-tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(2-chloro-5-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(1-18)

To a solution of 23 (10 mg, 0.01 mmol) in MeCN (1 mL) at roomtemperature, NCS (10 mg, 0.07 mmol) was added slowly. The reaction wasallowed to stir for about 2 h at room temperature. The reaction wasquenched with water (5 mL) and extracted with EtOAc (5 mL×3). Thecombined organic layers were dried over anhydrous sodium sulfate,filtered and concentrated in vacuum. The residue was purified by columnchromatography (40% EtOAc/petroleum ether, R_(f)=0.2) to yield 1-18 (2mg, 14% yield) as a pale brown solid: melting point 103.7-105.0° C.; ¹HNMR (400 MHz, acetone-d₆) δ 1.46 (s, 18H), 5.31 (s, 2H), 5.35 (s, 2H),7.05 (s, 1H), 7.73 (s, 1H), 7.80 (s, 1H), 8.01 (s, 1H), 8.35 (s, 1H),8.60 (s, 1H), 8.74 (s, 1H), 10.94 (s, 1H) ppm; ¹³C NMR (DMSO-d₆, 100MHz) δ 28.00, 28.00, 28.00, 28.00, 28.04, 28.04, 51.50, 56.12, 69.00,83.00, 108.20, 110.04, 113.40, 115.40, 116.83, 117.40, 119.00, 119.20,119.58, 119.95, 121.80, 123.34, 124.04, 125.83, 126.50, 126.59, 131.14,131.62, 132.43, 133.16, 142.33, 142.41, 155.48, 157.51, 166.53, 166.53,179.98, 181.85 ppm; HRMS ESI (M+Na⁺) calculated for C₃₈H₃₂Cl₆N₈NaO₈961.0372. found 961.0388; IR (KBr) 3442, 2979, 2920, 2851, 1744, 1629,1460, 1427, 1383, 1243, 1155, 1025, 752, 666 cm⁻¹. HPLC purity, 96.2%(Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=75:25; t_(R)=5.9 min).

2,2′-(4,4′-((4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(2-chloro-5-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diaceticacid (1-19)

To a solution of 1-18 (30 mg, 0.03 mmol) in anhydrous CH₂Cl₂ (2 mL),CF₃COOH (2 mL) was slowly added via a syringe at 0° C. The reaction wasallowed to warm up to room temperature and stirred for about 4 h. Thereaction was concentrated in vacuum. The residue was purified byreverse-phase column chromatography (C18 reverse silica gel, 6% AcOH,30% H₂O, 64% MeOH, R_(f)=0.2) to yield 1-19 (17 mg, 65% yield) as a palebrown solid: melting point 261.4-262.7° C.; ¹H NMR (400 MHz, CD₃OD) δ5.18 (s, 2H), 5.21 (s, 2H), 6.73 (s, 1H), 7.23 (s, 1H), 7.35 (s, 1H),7.48 (s, 1H), 8.59 (s, 1H), 8.87 (s, 1H), 8.48 (s, 1H), 8.57 (s, 1H)ppm; ¹³C NMR (DMSO-d₆, 100 MHz) δ 52.98, 53.62, 108.07, 109.84, 116.57,118.82, 119.32, 119.62, 121.71, 123.24, 123.81, 125.44, 125.90, 126.23,129.42, 131.17, 131.66, 131.68, 132.88, 133.50, 141.85, 141.97, 155.58,157.52, 161.00, 162.77, 169.56, 172.54, 179.98, 182.01 ppm; HRMS ESI(M+H⁺) calculated for C₃₀H₁₇Cl₆N₈O₈ 826.9301. found 826.9308; IR (KBr)3392, 2956, 2921, 2851, 1753, 1626, 1462, 1432, 1380, 1246, 1188, 1081,1025, 770 cm⁻¹. HPLC purity, 98.8% (Flow rate: 1.0 mL/min; Column:Waters C18, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25°C.; Mobile phase: MeOH:H₂O=65:35; t_(R)=4.2 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((4-(1-benzyl-1H-1,2,3-triazol-5-yl)-2-hydroxyphenyl)methanone)(1-20)

Under N₂, a mixture of 1-3 (20 mg, 0.04 mmol), (azidomethyl)benzene (29mg, 0.22 mmol), and CuCl (4 mg, 0.04 mmol) was dissolved in THF (5 mL).The reaction was allowed to warm up to reflux and stirred for about 8 h.The suspension was filtered and the filtrate was concentrated in vacuum.The residue was purified by column chromatography (5% acetone, 31%EtOAc, 64% petroleum ether, R_(f)=0.3) to yield 1-20 (17 mg, 55% yield)as a yellow solid: melting point 116.5-118.0° C.; ¹H NMR (400 MHz,CDCl₃) δ 5.56 (s, 2H), 5.57 (s, 2H), 6.16 (s, 1H), 7.01 (d, J=8.4 Hz,1H), 7.28-7.31 (m, 7H), 7.32-7.40 (m, 7H), 7.51 (br s, 1H), 7.69 (s,1H), 7.75 (s, 1H), 10.35 (br s, 1H), 10.83 (s, 1H), 11.39 (s, 1H) ppm;¹³C NMR (CDCl₃, 100 MHz) δ 54.36, 54.36, 112.01, 114.28, 114.28, 115.89,116.54, 117.18, 118.47, 118.65, 121.17, 121.32, 121.74, 121.94, 124.33,124.84, 128.12, 128.12, 128.12, 128.19, 128.19, 128.19, 128.95, 128.95,128.95, 129.22, 129.22, 129.22, 129.22, 131.25, 134.16, 134.16, 134.16,137.75, 138.85, 146.47, 162.06, 163.05, 185.56, 187.37 ppm; HRMS ESI(M+H⁺) calculated for C₄₀H₂₇Cl₄N₈O₄ 823.0909. found 823.0903; IR (KBr)2955, 2919, 2850, 1717, 1628, 1592, 1457, 1228, 1047, 935, 912, 790, 699cm⁻¹. HPLC purity, 98.5% (Flow rate: 1.0 mL/min; Column, Agilent ZORBAX300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=85:15; t_(R)=12.5 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-hydroxy-4-(1-phenyl-1H-1,2,3-triazol-5-yl)phenyl)methanone)(1-21)

Under N₂, a mixture of 1-3 (20 mg, 0.04 mmol), azidobenzene (26 mg, 0.22mmol), and CuCl (4 mg, 0.04 mmol) was dissolved in THF (5 mL). Thereaction was allowed to warm up to reflux and stirred for about 8 h. Thesuspension was filtered and the filtrate was concentrated in vacuum. Theresidue was purified by column chromatography (5% acetone, 31% EtOAc,64% petroleum ether, R_(f)=0.3) to yield 1-21 (20 mg, 70% yield) as ayellow solid: melting point 128.7-130.3° C.; ¹H NMR (400 MHz, CDCl₃) δ6.24 (s, 1H), 7.16 (d, J=8.0 Hz, 1H), 7.41-7.48 (m, 6H), 7.52-7.56 (m,5H), 7.77 (d, J=8.0 Hz, 4H), 8.24 (s, 1H), 8.30 (s, 1H), 10.35 (br s,1H), 10.91 (s, 1H), 11.47 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ109.13, 112.09, 114.48, 114.52, 116.00, 116.69, 117.17, 118.68, 118.83,119.23, 119.40, 120.51, 120.51, 120.51, 120.51, 120.51, 120.51, 121.76,122.02, 124.43, 124.86, 129.05, 129.09, 129.83, 129.83, 129.83, 129.83,129.83, 131.29, 134.25, 136.64, 137.44, 138.55, 146.68, 162.15, 163.12,185.54, 187.50 ppm; HRMS ESI (M+H⁺) calculated for C₃₈H₂₃Cl₄N₈O₄795.0596. found 795.0590; IR (KBr) 2955, 2918, 2849, 1701, 1630, 1594,1503, 1459, 1238, 1036, 935, 912, 757, 686 cm⁻¹. HPLC purity, 97.4%(Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm, 150×4.6mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=85:15; t_(R)=10.8 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((4-(1-cyclohexyl-1H-1,2,3-triazol-5-yl)-2-hydroxyphenyl)methanone)(1-22)

Under N₂, a mixture of 1-3 (20 mg, 0.04 mmol), azidocyclohexane (27 mg,0.22 mmol), and CuCl (4 mg, 0.04 mmol) was dissolved in THF (5 mL). Thereaction was allowed to warm up to reflux and stirred for about 8 h. Thesuspension was filtered and the filtrate was concentrated in vacuum. Theresidue was purified by column chromatography (5% acetone, 31% EtOAc,64% petroleum ether, R_(f)=0.3) to yield 1-22 (15 mg, 52% yield) as ayellow solid: melting point 143.8-145.0° C.; ¹H NMR (400 MHz, CDCl₃) δ1.25-1.39 (m, 2H), 1.44-1.59 (m, 4H), 1.78-1.87 (m, 6H), 1.94-1.97 (m,4H), 2.25-2.27 (m, 4H), 4.49 (t, J=10.0 Hz, 2H), 6.21 (s, 1H), 7.06 (d,J=8.4 Hz, 1H), 7.31-7.37 (m, 4H), 7.51 (br s, 1H), 7.82 (s, 1H), 7.87(s, 1H), 10.47 (br s, 1H), 10.91 (s, 1H), 11.44 (s, 1H) ppm; ¹³C NMR(CDCl₃, 100 MHz) δ 25.04, 25.04, 25.11, 25.11, 29.20, 29.65, 33.52,33.52, 33.52, 33.52, 60.36, 60.36, 109.00, 112.00, 114.12, 114.22,115.88, 116.54, 117.09, 118.32, 118.54, 119.10, 119.20, 121.64, 121.86,124.26, 124.86, 131.25, 134.18, 134.18, 138.22, 139.29, 145.60, 145.60,162.23, 163.12, 185.59, 187.39 ppm; HRMS ESI (M+H⁺) calculated forC₃₈H₃₅Cl₄N₈O₈O₄ 807.1535. found 807.1532; IR (KBr) 3129, 2923, 2853,1718, 1628, 1593, 1448, 1413, 1392, 1334, 1297, 1228, 1052, 914, 790cm⁻¹. HPLC purity, 95.0% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX300SB-C8, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.;Mobile phase: MeOH:H₂O=85:15; t_(R)=20.9 min).

(4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-diyl)bis((2-hydroxy-4-(1-octyl-1H-1,2,3-triazol-5-yl)phenyl)methanone)(1-23)

Under N₂, a mixture of 1-3 (20 mg, 0.04 mmol), 1-azidooctane (27 mg,0.22 mmol), and CuCl (4 mg, 0.04 mmol) was dissolved in THF (5 mL). Thereaction was allowed to warm up to reflux and stirred for about 8 h. Thesuspension was filtered and the filtrate was concentrated in vacuum. Theresidue was purified by column chromatography (5% acetone, 31% EtOAc,64% petroleum ether, R_(f)=0.3) to yield 1-23 (15 mg, 48% yield) as ayellow solid: melting point 85.2-86.7° C.; ¹H NMR (400 MHz, CDCl₃) δ0.84-0.91 (m, 6H), 1.14-1.35 (m, 20H), 1.93-1.95 (m, 4H), 4.40 (t, J=6.4Hz, 4H), 6.20 (s, 1H), 7.06 (d, J=8.4 Hz, 1H), 7.27-7.37 (m, 4H), 7.52(br s, 1H), 7.80 (s, 1H), 7.85 (s, 1H), 10.48 (br s, 1H), 10.87 (s, 1H),11.44 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz) δ 22.55, 22.55, 26.44,26.44, 28.91, 28.91, 28.91, 29.00, 29.00, 29.00, 30.23, 30.26, 31.65,31.65, 50.60, 50.60, 109.00, 112.02, 114.22, 114.26, 115.89, 116.56,117.13, 118.41, 118.61, 121.12, 121.25, 121.70, 122.00, 124.34, 124.87,126.85, 131.27, 134.20, 137.99, 139.09, 146.00, 162.13, 163.10, 163.10,185.59, 187.40 ppm; HRMS ESI (M+H⁺) calculated for C₄₂H₄₇Cl₄N₈O₄867.2474. found 867.2482; IR (KBr) 3392, 3080, 2955, 2922, 2852, 1629,1587, 1458, 1439, 1336, 1217, 1031, 914, 753, 703 cm⁻¹. HPLC purity,99.7% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 μm,150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=90:10; t_(R)=10.1 min).

Diethyl2,2′-(4,4′-((4,4′,5,5′-tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(1-24)

Under N₂, a mixture of 1-3 (20 mg, 0.04 mmol), ethyl 2-azidoacetate (28mg, 0.22 mmol), and CuCl (4 mg, 0.04 mmol) was dissolved in THF (5 mL).The reaction was allowed to warm up to reflux and stirred for about 4 h.The suspension was filtered and the filtrate was concentrated in vacuum.The residue was purified by column chromatography (5% acetone, 31%EtOAc, 64% petroleum ether, R_(f)=0.3) to yield 1-24 (15 mg, 52% yield)as a yellow solid: melting point 101.9-103.7° C.; ¹H NMR (400 MHz,acetone-d₆) δ 1.21-1.29 (m, 6H), 4.19-1.26 (m, 4H), 5.39 (s, 4H), 6.16(s, 1H), 7.23 (dd, J=8.4, 2.0 Hz, 1H), 7.33 (d, J=1.6 Hz, 1H), 7.39 (d,J=8.0 Hz, 1H), 7.42 (d, J=1.6 Hz, 1H), 7.92 (d, J=8.4 Hz, 1H), 8.20 (d,J=8.0 Hz, 1H), 8.39 (s, 1H), 8.49 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100 MHz)δ 14.04, 14.04, 50.97, 50.97, 62.62, 62.62, 109.05, 112.01, 114.38,114.38, 114.38, 115.96, 116.62, 117.08, 118.61, 118.73, 121.67, 121.90,122.73, 122.87, 124.37, 124.83, 131.25, 134.22, 137.46, 138.60, 146.48,162.05, 163.03, 166.12, 166.18, 185.58, 187.49, 187.49 ppm; HRMS ESI(M+H⁺) calculated for C₃₄H₂₇Cl₄N₈O₈ 815.0706. found 815.0707; IR (KBr)3670, 3389, 3080, 2959, 2920, 2850, 1752, 1631, 1584, 1481, 1439, 1216,1030, 753 cm⁻¹. HPLC purity, 98.4% (Flow rate: 1.0 mL/min; Column:Agilent ZORBAX 300SB-C8, 5 m, 150×4.6 mm; Wavelength: UV 254 nm;Temperature: 25° C.; Mobile phase: MeOH:H₂O=75:25; t_(R)=7.7 min).

Di-tert-butyl2,2′-(4,4′-((4,4′,5,5′-tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diacetate(1-25)

Under N₂, a mixture of 1-3 (10 mg, 0.02 mmol), tert-butyl 2-azidoacetate(14 mg, 0.11 mmol), and CuCl (2 mg, 0.02 mmol) was dissolved in THF (5mL). The reaction was allowed to warm up to reflux and stirred for about8 h. The suspension was filtered and the filtrate was concentrated invacuum. The residue was purified by column chromatography (5% acetone,31% EtOAc, 64% petroleum ether, R_(f)=0.3) to yield 1-25 (13 mg, 83%yield) as a yellow solid: melting point 138.3-140.0° C.; ¹H NMR (400MHz, acetone-d₆) δ 1.46 (s, 9H), 1.47 (s, 9H), 5.28 (s, 4H), 6.17 (s,1H), 7.23 (dd, J=8.4, 1.6 Hz, 1H), 7.34 (d, J=1.6 Hz, 1H), 7.40 (dd,J=8.4, 1.6 Hz, 1H), 7.43 (d, J=0.8 Hz, 1H), 7.91 (d, J=8.4 Hz, 1H), 8.18(d, J=8.0 Hz, 1H), 8.38 (s, 1H), 8.48 (s, 1H) ppm; ¹³C NMR (CDCl₃, 100MHz) δ 27.89, 27.89, 27.89, 27.89, 27.89, 27.89, 51.57, 51.57, 84.09,84.09, 108.98, 111.88, 114.23, 114.30, 115.90, 116.59, 116.98, 118.59,118.69, 121.66, 121.92, 122.74, 122.90, 122.90, 124.39, 124.82, 126.85,131.32, 134.19, 137.45, 138.65, 146.32, 161.95, 162.98, 165.10, 165.16,185.65, 187.45 ppm; HRMS ESI (M+H⁺) calculated for C₃₈H₃₅Cl₄N₈O₈871.1332. found 871.1325; IR (KBr) 3425, 3141, 2980, 2931, 1745, 1630,1599, 1454, 1369, 1239, 1156, 1048, 937, 852, 192 cm⁻¹. HPLC purity,99.5% (Flow rate: 1.0 mL/min; Column: Agilent ZORBAX 300SB-C8, 5 m,150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25° C.; Mobile phase:MeOH:H₂O=80:20; t_(R)=7.3 min).

2,2′-(4,4′-((4,4′,5,5′-Tetrachloro-1′H-1,3′-bipyrrole-2,2′-dicarbonyl)bis(3-hydroxy-4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))diaceticacid (1-26)

To a solution of 1-25 (17 mg, 0.02 mmol) in anhydrous CH₂Cl₂ (2 mL) wasslowly added CF₃COOH (2 mL) via a syringe at 0° C. The reaction wasallowed to warm up to room temperature and stirred for about 4 h andconcentrated in vacuum. The residue was purified by reverse-phase columnchromatography (C18 reverse silica gel, 6% AcOH, 30% H₂O, 64% MeOH,R_(f)=0.2) to yield 1-26 (14 mg, 94% yield) as a yellow solid: meltingpoint 100.3-102.0° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 4.80 (s, 2H), 4.83(s, 2H), 6.09 (s, 1H), 7.15 (d, J=8.4 Hz, 1H), 7.22 (s, 2H), 7.32 (s,1H), 7.53 (d, J=8.4 Hz, 1H), 7.97 (d, J=8.4 Hz, 1H), 8.38 (s, 1H), 8.41(s, 1H) ppm; ¹³C NMR (DMSO-d₆, 100 MHz) δ 53.17, 53.22, 109.12, 109.58,112.93, 114.34, 114.99, 116.28, 121.49, 122.71, 123.87, 124.51, 124.67,129.03, 130.14, 132.35, 133.28, 135.63, 137.40, 145.28, 145.79, 158.86,159.46, 169.77, 169.80, 172.88, 177.84, 182.18, 185.38, 190.74 ppm; HRMSESI (M+^(H)) calculated for C₃₀H₁₉Cl₄N₈O₈ 759.0080. found 759.0054; IR(KBr) 3417, 3268, 3136, 1627, 1457, 1431, 1393, 1307, 1234, 1025, 1002,936, 799, 688 cm⁻¹. HPLC purity, 99.7% (Flow rate: 1.0 mL/min; Column:Waters C18, 5 μm, 150×4.6 mm; Wavelength: UV 254 nm; Temperature: 25°C.; Mobile phase: MeOH:H₂O=65:35; t_(R)=4.1 min).

Enzyme-Linked Immunosorbent Assay (ELISA).

ELISAs were performed using a similar procedure as previously described(Doi K, et al. J. Biol. Chem. 2012, 287, 10224-10235). Briefly, 40 nM ofbiotinylated Bim BH3 peptide (Biomatik) in SuperBlock blocking buffer(Pierce) was incubated in high-binding capacity streptavidin-coatedplates (Pierce) for 2 h. Compounds were diluted in 120 μl of PBScontaining 10 nM of GST-Mcl-1 or GST-Bcl-x_(L) in 1.5 mL tubes for 15min. Wells were washed with wash buffer (PBS containing 0.05% Tween-20)and then 100 μL of the compound/GST-protein mixture was transferred tothe wells. The plates were incubated for 2 h, and then wells were washedwith wash buffer. HRP-conjugated anti-GST antibody (Bethyl Laboratories)was diluted 1:2000 in SuperBlock and 100 μL was transferred to eachwell. The plate was incubated for 1 h, and then wells were washed withwash buffer followed by PBS. 100 μL of SureBlue TMB Microwell PeroxidaseSubstrate (VWR) was added to each well and plates were developed for5-10 min. 100 μL of 1 N HCl was added to each well to stop the reactionand absorbance was read at 450 nm using a Quant plate reader (Bio-Tek).

Protein Expression and Purification.

Mcl-1 (residues 172-327) was expressed as maltose binding protein (MBP)fusions from the pSV282 vector (Fire E, et al. Protein Science. 2010,19, 507-519). Mcl-1 protein was expressed in BL21(DE3) Codon+ at 18° C.for 17 h. Cells were lysed using a high pressure homogenizer in 100 mMTris-HCl pH 8, 250 mM NaCl, 5 mM 2-mercaptoethanol, 25 mM imidazole, andComplete EDTA-free protease inhibitor cocktail, and purified byNi-affinity chromatography. Purified Mcl-1-MBP fusion protein wascleaved with TEV protease in 100 mM Tris-HCl pH 8, 60 mM Citrate, 5 mMBME at 4° C. for 17 h. Cleaved Mcl-1 was separated from MBP bysubtractive Ni-affinity chromatography followed by gel filtration of theflow through using a Superdex75 10/300 gel filtration column. Theuniformly ¹⁵N-labelled protein samples were prepared by growing thebacteria in minimal medium containing ¹⁵N-labeled NH₄Cl followed by thesame purification procedure.

NMR Spectroscopy.

¹⁵N-HSQC spectra were recorded on ¹⁵N-labelled Mcl-1 at 0.1 mM preparedin 50 mM potassium phosphate solution at pH 6.5, 50 mM NaCl and 1 mMDTT. Titrations up to 100 μM of 1-21 and 1-18 compounds were performed.NMR spectra were acquired at 25° C. on an Inova 600 MHz spectrometerequipped with a cryoprobe and analyzed in CCPNMR (Vranken W F, et al.Proteins. 2005, 59, 687-696).

Docking Studies.

NMR-guided docking of 1-21 into the X-ray crystal structure of Mcl-1(PDB ID: 3KJ0) was performed using Glide (Maestro, Schrodinger)(Friesner R A et al. J. Med. Chem. 2004, 47, 1739-1749; Friesner R A, etal. J. Med. Chem. 2006, 49, 6177-6196; Halgren T A, et al. J. Med. Chem.2004, 47, 1750-1759; Gavathiotis E, et al. Nat. Chem. Biol. 2012, 8,639-645). The lowest-energy docking pose is consistent with the observedNMR-chemical shift perturbation data. Pymol is used for preparing thehighlighted poses.

Results and Discussion

Optimization of marinopyrroles as either Mcl-1-selective,Bcl-x_(L)-selective or dual Mcl-1 and Bcl-x_(L) antagonists will lead tovaluable chemical probes and eventually to natural product-basedanti-cancer agents. As shown in Table 2, marinopyrroles have at leasteight sites amenable for optimization to accomplish the desired activityand selectivity. As discussed in disruptor design (vide supra),optimization includes substitutions on the pyrrole nitrogen, two phenylrings, two carbonyl groups, replacing rings A and B, substitution atC-5′ position, intramolecular cyclization between 2-OH and chloro onC-5′, and substitution on the second pyrrole ring.

Mcl-1/Bim Disruption SAR.

The potency of the parent natural product Marinopyrrole A [(−)-1] toinhibit the binding of Mcl-1 to Bim was moderate (IC₅₀=12.5 μM) whilethe atropisomer (+)-1 exhibited similar potentcy (IC₅₀=12.7 μM), and thesynthetic marinopyrrole A [(±)-1] displayed an IC₅₀ value of 8.9 μM.From the structure-activity relationship (SAR) studies of thesymmetrical derivatives of (±)-1, the following overall conclusions canbe drawn. Whereas, analogues of (±)-1 where the 4 and 4′ hydrogens ofphenyl rings A and B, respectively, were replaced by alkyls, alkenes andalkynes such as 1-2 to 1-5 gained some potency (up to 4-fold), thoseanalogues where the 4- and 4′-hydrogens were replaced by sulfide- orbistriazole-containing moieties such as 1-10 and 1-26 gained substantialpotency (up to 15-fold) (Table 2). Finally, analogues with hydroxyl- andcarboxylate-containing groups at the 4 and 4′ positions such as 1-7 and1-16 lost potency, but neutralizing the negative charge of thecarboxylate by alkylation improves inhibitory activity (compare 1-16 to1-12, 1-15 to 1-9, 1-26 to 1-25, 1-19 to 1-18).

In the sulfide-containing series, derivatives with S-benzyl (1-10) orSCH₂(p-MeOPh) (1-11) were the most potent with IC₅₀ values of 700 nM(Table 2). In this series, replacing the phenyl in 1-10 (IC₅₀=700 nM) bythe negatively-charged carboxylate as in 1-15 (6100 nM) lead to an8.7-fold loss in potency to disrupt Mcl-1/Bim binding. However,neutralization of the 1-15 negative charge as in the ethylesterderivative 1-9 improved the potency by 3.4-fold from 6100 nM to 1800 nM.This suggests that substituents at the 4- and 4′-positions of thesulfide-containing marinopyrroles bind to the Mcl-1 BH3 binding pocketin the vicinity of a negatively charged environment. Furthermore,replacing the sulfide by the corresponding sulphone consistentlyresulted in substantial (up 25-fold) loss of potency [compare 1-10 to1-13 (16-fold loss), 1-11 to 1-14 (25-fold loss), 1-15 to 1-16 (10-foldloss) and 1-9 to 1-12 (21-fold loss)](Table 2). This loss of potencysuggests that the BH3 binding pocket prefers the molecular geometry ofthe —S— over that of the —SO₂— groups.

In the bistriazole series, the octyl-triazole-containing analogue 1-23was the most potent with an IC₅₀ value of 600 nM (Table 2). Replacingthe octyl in 1-23 with cyclohexyl (1-22), phenyl (1-21) or benzyl (1-20)resulted in 2.3-, 2.5- and 5.5-fold loss of potency, respectively,suggesting that the extended aliphatic octyl is better accommodated thanthe cyclohexyl or phenyl in the BH3 binding domain in Mcl-1. Similarly,replacing the octyl with a methylene carboxylate (1-26), itscorresponding ethyl (1-24) or t-butyl (1-25) esters also resulted inloss of potency. However, addition of chloro group to the 5- and5′-positions to 1-26, 1-24 and 1-25 improves their potencies [compare1-26 (16.5 μM) to 1-19 (5.2 μM); 1-24 (18.4 μM) to 1-17 (7.8 μM); 1-25(5.1 μM) to 1-18 (1.6 μM)]. Interestingly, the same chloro modificationson the 5- and 5′-positions also enhanced the potency of marinopyrrolesanalogues that do not contain a triazole such as 1-7 (39.5 μM) to 1-8(10.7 μM), suggesting that the chloro groups in those positions occupy aregion in the BH3 binding domain of Mcl-1 that increases the affinity.

Another potent analogue of (±)-1 (IC₅₀=8.9 μM) is 1-6 (IC₅₀=1 μM) wherethe hydrogens at 4- and 4′-positions were replaced bytrifluoromethanesulfonate groups (Table 2). However, replacing thesehydrogens with trifluoromethyl groups as in 1-1 had little effect onpotency (IC₅₀=8.1 μM). Substituting the 4- and 4′-hydrogens of (±)-1with ethyl as in 1-5 (2.1 μM), CH═CH₂ as in 1-4 (3.7 μM) or ethyne as in1-3 (3.7 μM) enhanced potency by 2- to 4-fold suggesting that this areaof the marinopyrroles binds a hydrophobic pocket in BH3 binding domainof Mcl-1.

In the non-symmetrical marinopyrroles, replacing the 3′-, 4′- or5′-hydrogens in (±)-1 with chloro groups as in 1-28, 1-30 or 1-29resulted in a slight increase in potency (3.9 to 6.5 M). Similarsubstitutions with fluoro groups as in 1-31 to 1-33 did not enhancepotency. N-methylation of (±)-1 as in 1-35 was not tolerated (IC₅₀>100μM). Similarly, the N-methyl analogue of (±)-1 where the 2- and2′-hydroxyl groups were replaced by 2- and 2′-methoxy groups also lostpotency but only by 2-fold (IC₅₀=15.5 μM). This suggested that the NHgroup of marinopyrroles is involved in H-bonding interactions at the BH3binding domain. Replacing phenol A but not phenol B of (±)-1 with anethoxy carbonyl group as in 1-37 and 1-36, respectively, resulted in a3-fold loss of activity (Table 2).

Bcl-x_(L)/Bim Disruption SAR.

While the (±)-1 was less potent at inhibiting Bcl-x_(L)/Bim (IC₅₀=16.4μM) than Mcl-1/Bim binding (IC₅₀=8.9 μM), the SAR generated was similar,except for several important differences. As with Mcl-1/Bim SAR, some ofthe symmetrical derivatives of (∓)-1 where the 4- and 4′-hydrogens werereplaced by sulfide-containing moieties gained a significant amount ofpotency (up to 23 fold). For example, derivatives with S-benzyl (1-10)or SCH₂(p-MeOPh) (1-11) were the most potent with IC₅₀ values of 600 nM(Table 2). Replacing the phenyl in 1-10 or the para-methoxy-phenyl in1-11 by the negatively-charged carboxylate as in 1-15 was not tolerated(IC₅₀>100 μM). However, neutralization of the 1-15 negative charge as inthe ethylester derivative 1-9 (IC₅₀=1200 nM) greatly enhanced thepotency by over 83-fold. Another substitution that was not tolerated isthe replacement of the sulfide by sulphone spacer (compare 1-10 to 1-13,1-11 to 1-14, 1-15 to 1-16 and 1-9 to 1-12). As was the case forMcl-1/Bim, among the bistriazole-containing analogues, theoctyl-triazole-containing analogue 1-23 was the most potent to inhibitBcl-x_(L)/Bim binding [IC₅₀=500 nM; 32.8-fold more potent than (±)-1(Table 2)]. However, unlike in Mcl-1/Bim, replacing the octyl in 1-23with phenyl 1-21 (IC₅₀=800 nM) or benzyl 1-20 (IC=1600 nM) were betterthan with cyclohexyl 1-22 (IC₅₀=3100 nM) (Table 2). Replacing the octylwith a methylene carboxylate (1-26) and its corresponding ethyl ester(1-24) were not tolerated. In contrast to Mcl-1/Bim, addition of chlorogroups to the 5- and 5′-positions to 1-26, 1-24 and 1-25 as in 1-19,1-17 and 1-18, respectively, did not improve their potencies, neitherdid it enhance the potency of marinopyrroles analogues that do notcontain a triazole (compare 1-7 to 1-8). Hydrophobic substitutions atthe 4- and 4′-positions of (±)-1 with trifluoromethanesulfonate groupsas in 1-6, ethyl as in 1-5, ethyl alkene as in 1-4, ethyne as in 1-3 andCF₃ as in 1-1 all moderately improved potency with 1-6 being the mostpotent (Table 2).

In the non-symmetrical marinopyrroles, replacing the 3′- and 4′-but notthe 5′-hydrogens in (±)-1 with chloro groups as in 1-28, 1-30 and 1-29,respectively, resulted in a slight increase in potency. Similarsubstitutions with fluoro groups as in 1-31 to 1-33 decreased potency.Unlike in Mcl-1/Bim where it was not tolerated, N-methylation of (±)-1as in 1-35 (IC₅₀=7.1 M) increase potency of (±)-1 (IC₅₀=16.4 μM) toinhibit Bcl-x_(L)/Bim binding. In contrast, the N-methyl analogue of(±)-1 where the 2- and 2′-hydroxyl groups were replaced by 2- and2′-methoxy groups lost 4-fold potency (IC₅₀=64.9 μM). Finally, replacingphenol A but not phenol B of (±)-1 with an ethoxy carbonyl group as in1-37 and 1-36, respectively, resulted in a 6-fold loss of activity(Table 2).

Mcl-1-Selective, Bcl-x_(L)-Selective and Dual Mcl-1/Bcl-x_(L)Marinopyrrole Antagonists

From the SAR results several selective Mcl-1/Bim and Bcl-x_(L)/Bimdisruptors emerged. The most selective Mcl-1 antagonist was 1-15 withover 16-fold more selectivity for disrupting Mcl-1/Bim overBcl-x_(L)/Bim binding. However, neutralizing the carboxylate negativecharge with an ethyl ester as in 1-9 or replacing the carboxylate witheither a phenyl (1-10) or a methoxyphenol (1-11) not only greatlyincreased the potency as discussed above, but also reversed theselectivity resulting in some of the most potent Mcl-1 and Bcl-x_(L)dual inhibitors, with 1-9 being slightly more potent against Bcl-x_(L)(Table 2). In the triazole series, 1-20 with a benzyl and 1-21 with aphenyl were 2-fold more selective for Bcl-x_(L) over Mcl-1. Thisselectivity was reversed towards Mcl-1 when the benzyl or phenyl werereplaced with ethyl carboxylate as in 1-24 (5.4 fold), carboxylate as in1-26 (3.0 fold), cyclohexyl as in 1-22 (2.8 fold) or t-butyl carboxylateas in 1-25 (1.6 fold). Replacement with an octyl group as in 1-23resulted in one of the most potent dual Mcl-1 and Bcl-x_(L) antagonists.In the triazole series adding chloro groups to the 5- and 5′-positionsincreases selectivity for Mcl-1 over Bcl-x_(L) [compare 1-17 (12.9 fold)to 1-24 (5.4 fold), 1-19 (9.6 fold) to 1-26 (3 fold) and 1-18 (8.3 fold)to 1-25 (1.6 fold)].

Substituting the 4- and 4′-hydrogens in (±)-1 with hydrophobic groupssuch as ethyl alkene (1-4), methyl (1-2), ethyne (1-3), ethyl (1-5) andCF₃ (1-1) all lead to dual Mcl-1 and Bcl-x_(L) antagonists (Table 2). Anumber of non-symmetrical analogues were also dual Mcl-1 and BclxLantagonists. These include 1-27, 1-30, 1-31, and others (Table 2). Themost selective Bcl-x_(L) antagonist is 1-35 with 12.7-fold selectivityfor Bcl-x_(L) over Mcl-1. Replacing the 2- and 2′-hydroxyl groups in1-35 with 2- and 2′-methoxy groups as in 1-34 leads to full reversal toa Mcl-1 selective antagonist (4.2 fold), suggesting the key roles thatthe hydroxyl groups play in the binding of 1-35 to the BH3 domain ofBcl-x_(L). Furthermore, replacing the N-methyl in 1-35 with a hydrogenas in (±)-1 also leads to reversal of selectivity for Mcl-1 (1.8 fold)suggesting the importance of the N-hydrogen in binding to Mcl-1 but notBcl-x_(L).

TABLE 2-A ELISA results and physicochemical property of marinopyrroles.Mcl- Bcl- Clog ID R¹ R² 1/Bim^(a) x_(L)/Bim^(a) p^(b) (±)-1^(c) 2-OH2′-OH  8.9 ± 1.0 16.4 ± 3.3 5.6 (−)-1^(c) 2-OH 2′-OH 12.5 ± 1.4 12.0 ±2.8 5.6 (+)-1^(c) 2-OH 2′-OH 12.7 ± 1.0 19.7 ± 3.6 5.6 1-17

 7.8 ± 1.5 >100 5.9 1-18

 1.6 ± 0.6 14.0 ± 4.7 6.9 1-19

 5.2 ± 0.8  >50 5.1 1-20

 3.3 ± 0.9  1.6 ± 0.3 8.6 1-21

 1.5 ± 0.2  0.8 ± 0.2 8.4 1-22

 1.4 ± 0.5  3.8 ± 1.3 8.2 1-23

 0.6 ± 0.3  0.5 ± 0.1 10.6 1-24

18.4 ± 0.3 >100 4.9 1-25

 5.1 ± 0.4  8.1 ± 2.9 5.8 1-26

16.5 ± 1.9  >50 4.1 ^(a)IC₅₀ in μM (average ± SEM, n ≧ 3) unlessspecified; ^(b)Calculated using ChemAxon; ^(c)Activity as disruptors ofMcl-1 and Bcl-x_(L), reported previously and here for SAR discussion(Cheng C, et al. Submitted to Mar. Drugs);

TABLE 2-B ELISA results and physicochemical property of marinopyrroles.Mcl- Bcl- ID R¹ R² 1/Bim^(a) x_(L)/Bim^(a) Clog p^(b) (±)-1^(c) 2-OH2′-OH 8.9 ± 1.0 16.4 ± 3.3  5.6 (−)-1^(c) 2-OH 2′-OH 12.5 ± 1.4  12.0 ±2.8  5.6 (+)-1^(c) 2-OH 2′-OH 12.7 ± 1.0  19.7 ± 3.6  5.6 1-1^(d)2-OH-4-CF₃ 2′-OH-4′-CF₃ 8.1 ± 0.9 9.7 ± 1.3 7.3 1-2 2-OH-5-Cl-4-Me2′-OH-5′-Cl-4′-Me 2.6 ± 0.6 2.5 ± 0.4 7.5 1-3 2-OH-4-C≡CH 2′-OH-4′-C≡CH3.9 ± 0.2 5.6 ± 0.5 5.9 1-4 2-OH-4-CH═CH₂ 2′-OH-4′-CH═CH₂ 3.7 ± 0.5 3.5± 0.7 7.4 1-5 2-OH-4-Et 2′-OH-4′-Et 2.1 ± 0.5 3.9 ± 1.2 7.3 1-6^(d)2-OH-4-OSO₂CF₃ 2′-OH-4′-OSO₂CF₃ 1.0 ± 0.3 2.5 ± 0.7 8.1 1-7^(d)2-OH-4-OH 2′-OH-4′-OH 39.5 ± 6.2   >50 5.0 1-8^(d) 2-OH-5-Cl-4-OH2′-OH-5′-Cl-4′-OH 10.7 ± 0.2   >50 6.0 1-9 2-OH-4-SCH₂CO₂Et2′-OH-4′-SCH₂CO₂Et 1.8 ± 0.3 1.2 ± 0.2 6.1 1-10 2-OH-4-SCH₂Ph2′-OH-4′-SCH₂Ph 0.7 ± 0.2 0.6 ± 0.2 10.2 1-11 2-OH-4-SCH₂(p-2′-OH-4′-SCH₂(p- 0.7 ± 0.1 0.6 ± 0.1 9.7 MeOPh) MeOPh) 1-122-OH-4-SO₂CH₂CO₂Et 2′-OH-4′-SO₂CH₂CO₂Et 37.3 ± 3.1   >50 3.7 1-132-OH-4-SO₂CH₂Ph 2′-OH-4′-SO₂CH₂Ph 11.2 ± 4.0  69.3 ± 15.8 6.9 1-142-OH-4-SO₂CH₂(p- 2′-OH-4′-SO₂CH₂(p- 17.4 ± 3.1  >100 6.4 MeOPh) MeOPh)1-15 2-OH-4-SCH₂CO₂H 2′-OH-4′-SCH₂CO₂H 6.1 ± 1.3 >100 5.3 1-162-OH-4-SO₂CH₂CO₂H 2′-OH-4′-SO₂CH₂CO₂H 63.0 ± 5.4  >100 2.9 1-27^(e)2-OMe 2′-OMe-4′-Cl 8.0 ± 1.6 9.5 ± 2.2 4.9 1-28^(e) 2-OH 2′-OH-3′-Cl 4.1± 1.4 10.1 ± 2.2  6.1 1-29^(e) 2-OH 2′-OH-5′-Cl 3.9 ± 1.1 18.3 ± 3.0 6.1 1-30^(e) 2-OH 2′-OH-4′-Cl 6.5 ± 1.3 9.2 ± 2.3 6.1 1-31^(f) 2-OH2′-OH-5′-F 8.9 ± 0.9 13.3 ± 3.3  5.7 1-32^(f) 2-OH 2′-OH-4′-F 9.6 ± 0.421.3 ± 5.6  5.7 1-33^(f) 2-OH 2′-OH-6′-F 13.1 ± 0.3  45.7 ± 10.0 5.71-34^(g) 2-OMe 2′-OMe 15.5 ± 3.3  64.9 ± 15.5 4.6 1-35^(g) 2-OH2′-OH >100 7.1 ± 2.1 5.8 1-36^(h) CO₂Et replacing COAr 2′-OH 11.5 ± 1.9 17.6 ± 4.5  4.5 1-37^(h) 2-OH CO₂Et replacing COAr 25.1 ± 4.7     96.64.5 ABT-263    4.3 ± 0.4 nM ^(a)IC₅₀ in μM (average ± SEM, n ≧ 3) unlessspecified; ^(b)Calculated using ChemAxon; ^(c)Activity as disruptors ofMcl-1 and Bcl-x_(L) reported previously and here for SAR discussion(Cheng C, et al. Submitted to Mar. Drugs); ^(d)Chemistry, anti-MRSAactivity and Clog p were reported previously (Cheng C, et al. Mar.Drugs. 2013, 11, 2927-2948); ^(e)Chemistry and anti-MRSA activity werereported previously (Liu Y, et al. Mar. Drugs. 2012, 10, 953-962);^(f)Chemistry and anti-MRSA activity were reported previously (Liu Y, etal. Submitted to Mar. Drugs); ^(g)N—Me analogue; ^(h)Compounds reportedpreviously (Nicolaou K C, et al. Tetrahedron Lett. 2011, 52, 2041-2043).

CONCLUSIONS

Described herein is the design, synthesis and SAR studies ofmarinopyrrole derivatives. Structural characterization of marinopyrrolebinding to Mcl-1 suggests that bistriazole marinopyrrole 1-21 occupiesthe p1-p4 pockets along α-helical Bim peptide by NMR chemical shiftperturbations assisted with molecular modeling. Comprehensive SARstudies demonstrated: i) symmetrical marinopyrroles with hydrophobicsubstituents in the para-position to the carbonyl group are desired foractivity against Mcl-1/Bim and Bcl-x_(L)/Bim but hydrophilicsubstituents are not tolerated; ii) substituents with sulfide spacerslead to some of the most potent disruptors of Mcl-1/Bim andBcl-x_(L)/Bim binding, but those with sulfone spacers are tolerated;iii) substituents with triazole spacer containing octyl, cyclohexyl andphenyl are most potent against both Mcl-1 and Bcl-x_(L), but thosecontaining carboxylates such as in 1-17, 1-19, 1-24 and 1-26 are notactive against Bcl-x_(L)/Bim; iv) substituents with chlorine or fluorinein B ring of non-symmetrical marinopyrroles are tolerated in most cases;v) N-methylation of marinopyrrole A (1-36) leads to high selectivity forBcl-x_(L)/Bim over Mcl-/Bim whereas the sulfide 1-15 was much moreselective for Mcl-1 over Bcl-x_(L); vi) replacing A ring with CO₂Et isalso allowed.

Example 4

Previous reports have discussed the ability of marinopyrrole A toinhibit the binding of Mcl-1 to Bim. However, marinopyrrole A onlymoderately inhibits the binding to Mcl-1 to Bim (8.9 μM), itsselectivity for Mcl-1 over Bcl-x_(L) is only two fold, and it suffersfrom poor solubility. Herein, marinopyrrole A analogues were synthesizedwhich can: improve solubility and potency, identify chemical probesselective for Mcl-1, Bcl-x_(L) or Bcl-2, and be developed as anti-cancerdrugs. The molecular geometry of marinopyrrole A offers excellentopportunities to reach these goals by decorating this natural productbased bispyrrole system with a large number of diverse functionalgroups. Marinopyrrole has at least eight sites amenable to optimizationto accomplish the desired activity and selectivity.

Results and Discussion

Structure activity relationship studies using Mcl-1/Bim andBcl-x_(L)/Bim ELISA assays were used to identify marinopyrrole Aanalogues that are Mcl-1-selective and Bcl-x_(L)-selective antagonistsas well as dual inhibitors of Bim binding to both Mcl-1 and Bcl-x_(L).The parent marinopyrrole A [1=(±)−(1)] inhibits the binding of Mcl-1 toBim with only moderate potency (IC₅₀=8.9 μM) (Table 3). All datareported in this example were performed with both Mcl-1 and Bcl-xL at 10nM. Table 3 shows that substitutions of the para-hydrogens relative tothe carbonyl group on both rings A and B in 1 with sulfide- orbistriazole-containing moieties such as I-13 and II-11 resulted in themost potent analogues (500 nM; up to 32-fold more potent than 1).Derivatives with alkyls, alkenes, alkynes and trifluoromethanesulphonateat the para positions such as I-2 to I-6, respectively, gained moderatepotency (up to nine-fold). The most selective Mcl-1 antagonist wassulfide-containing 1-10 with over 16-fold more selectivity fordisrupting Mcl-1/Bim over Bcl-xL/Bim binding, whereas the most selectiveBcl-xL antagonist was VI-12 (N-methyl-1) with 12.7-fold selectivity forBcl-xL over Mcl-1. The most potent dual Mcl-1 and Bcl-xL antagonistswere sulfide 1-13 and triazole II-9 (Table 3).

TABLE 3-A ELISA results of selected marinopyrroles of structure I pK_(a)pK_(a) pK_(a) pK_(a) Clog ID R¹ R² Mcl-1-Bim^(a) Bcl-xL-Bim^(a) 1^(d)2^(d) 3^(d,e) 4^(d,e) p^(d) (±)-1 H H 8.9 ± 1.0 16.4 ± 3.3  7.8 8.4 — —5.6 (+)-1 H H 12.7 ± 1.0  19.7 ± 3.6  — — — — — (−)-1 H H 12.5 ± 1.4 12.0 ± 2.8  — — — — — I-1 H CF₃ 8.1 ± 0.9 9.7 ± 1.3 — — — — — I-2 HCH₂C≡CH 3.9 ± 0.2 5.6 ± 0.5 — — — — — I-3 H CH₂CH═CH₂ 3.7 ± 0.5 3.5 ±0.7 — — — — — I-4 Cl CH₃ 2.6 ± 0.6 2.5 ± 0.4 — — — — — I-5 H CH₂CH₃ 2.1± 0.5 3.9 ± 1.3 — — — — — I-6 H OSO₂CF₃ 1.0 ± 0.3 2.1 ± 0.7 — — — — —I-7 H OH 39.5 ± 6.2   >50 — — — — — I-8 Cl OH 10.7 ± 0.2   >50 — — — — —I-9 H SCH₂CO₂CH₂CH₃ 1.8 ± 0.3 1.2 ± 0.2 7.8 8.4 — — 6.1 I-10 H SCH₂CO₂H6.1 ± 1.3 >100 7.8 8.4 2.9 3.5 5.3 I-11 H SO₂CH₂CO₂H 63.0 ± 5.4  >1006.7 7.3 2.2 2.9 2.9 I-12 H SO₂CH₂CO₂CH₂CH₃ 37.3 ± 3.1  >100 6.7 7.3 — —3.7 I-13^(b) H SCH₂Ph 0.7 ± 0.2 0.6 ± 0.2 7.8 8.4 — — 10.2 I-14^(b) HSO₂CH₂Ph 7.3 ± 1.4 69.3 ± 5.8  6.7 7.3 — — 6.9 I-15^(b) HSCH₂-para-CH₃OPh 0.7 ± 0.1 0.6 ± 0.1 7.8 8.4 — — 9.7 I-16 HSO₂CH₂-para-CH₃OPh 17.4 ± 3.1  >100 6.7 7.3 — — 6.4 I-17 H CO₂CH₃ 16.9 ±2.3  >100 — — — — — I-18 H CO₂H 61.4 ± 7.6  >100 — — — — — I-19 HPO(OCH₂CH₃)₂ 7.7 ± 2.2 >100 — — — — — I-20 H PO(OH)₂ 10.9 ± 3.1  27.3 ±7.2  — — — — — I-21^(c) H H 4.5 ± 0.9 7.3 ± 0.9 — — — — — ^(a)IC₅₀(average ± SEM) in μM, n ≧ 3 ^(b)Ph = phenyl ^(c)tetra-Br replacingtetra-Cl in 1 ^(d)calculated using ChemAxon Software Version 5.12.3^(e)pK_(a) values frm carboxylic acid group

TABLE 3-B ELISA results of selected marinopyrroles of structure II ID R³R⁴ Mcl-1-Bim^(a) Bcl-xL-Bim^(a) II-2 Cl CH₂CO₂CH₂CH₃ 7.8 ± 1.5 >100II-3^(d) Cl CH₂CO₂CO₂Bu 1.6 ± 0.6 13.2 ± 3.9  II-4 Cl CH₂CO₂H 5.2 ± 0.8 >50 II-5 H CH₂CO₂CH₂CH₃ 18.4 ± 0.3  >100 II-6^(d) H CH₂CO₂Bu 5.1 ± 0.48.1 ± 2.5 II-7 H CH₂CO₂H 16.5 ± 1.9   >50 II-8^(b) H CH₂Ph 3.3 ± 0.9 1.6± 0.3 II-9^(b) H Ph 1.5 ± 0.2 0.8 ± 0.2 II-10 H cyclohexane 1.4 ± 0.53.8 ± 1.3 II-11 H n-octane 0.6 ± 0.3 0.5 ± 0.1 ^(a)IC₅₀ (average ± SEM)in μM, n ≧ 3 ^(b)Ph = phenyl ^(d)Bu = butyl

TABLE 3-C ELISA results of selected marinopyrroles of structure IVBcl-xL- pK_(a) pK_(a) pK_(a) pK_(a) Clog ID R⁶ R⁷ Mcl-1-Bim^(a) Bim^(a)1^(c) 2^(c) 3^(c,d) 4^(c,d) p^(c) IV-1 OH OCH₂CH₃ 25.1 ± 4.7  96.6 (n =2) — 8.1 — — 4.5 IV-2^(b) OCH₃ 4-Cl-2-OCH₃—Ph 8.0 ± 1.6 9.5 ± 2.2 — — —— — IV-3^(b) OH 3-Cl-2-OH—Ph 4.1 ± 1.4 10.1 ± 2.2  — — — — — IV-4^(b) OH5-Cl-2-OH—Ph 3.9 ± 1.1 18.3 ± 3.0  — — — — — IV-5^(b) OH 4-Cl-2-OH—Ph6.5 ± 1.3 9.2 ± 2.3 — — — — — IV-6^(b) OH 5-F-2-OH—Ph 8.9 ± 0.9 13.3 ±3.3  — — — — — IV-7^(b) OH 4-F-2-OH—Ph 9.6 ± 0.4 21.3 ± 5.6  — — — — —IV-8^(b) OH 6-F-2-OH—Ph 13.1 ± 0.3 43.7 ± 10.0 — — — — — ^(a)IC₅₀(average ± SEM) in 1 μM, n ≧ 3 ^(b)Ph = phenyl ^(c)calculated usingChemAxon Software Version 5.12.3 ^(d)pK_(a) values frm carboxylic acidgroup

TABLE 3-D ELISA results of selected marinopyrroles of structure V Mcl-1-Bcl-xL- pK_(a) pK_(a) pK_(a) pK_(a) Clog ID R⁸ R⁹ Bim^(a) Bim^(a) 1^(b)2^(b) 3^(b,c) 4^(b,c) p^(b) V-1 OH OCH₂CH₃ 11.5 ± 1.9 17.6 ± 4.5 8.1 — —— 4.5 ^(a)IC₅₀ (average ± SEM) in μM, n ≧ 3 ^(b)calculated usingChemAxon Software Version 5.12.3 ^(c)pK_(a) values frm carboxylic acidgroup

TABLE 3-E ELISA results of selected marinopyrroles of structure VI IDR¹⁰ R¹¹ R¹² R¹³ R Mcl-1-Bim^(a) Bcl-xL-Bim^(a) VI-1 OCH H H H H — — VI-2H OCH₃ H H H — — VI-3 H H OCH₃ H H — — VI-4 F H H H H — — VI-5 H F H H H— — VI-6 H H F H H — — VI-7 CF₃ H H H H — — VI-8 H H CF₃ H H — — VI-9 OHH OCH₃ Cl H — — VI-10 OCH₃ H OCH₃ Cl H — — VI-11 OCH₃ H H H CH₃ 15.5 ±3.3 64.9 ± 15.5 VI-12 OH H H H CH₃ >100 7.9 ± 1.8 ^(a)IC₅₀ (average ±SEM) in μM, n ≧ 3

Fluorescence Quenching Demonstrates Direct Binding.

To investigate direct binding of compounds to Mcl-1, afluorescence-quenching (FQ) assay based on the intrinsic Trpfluorescence of Mcl-1 was established. Using this assay, direct bindingof several marinopyrrole analogues to Mcl-1 was confirmed by generatingbinding isotherms and calculating the binding constants for II-4 andI-20 (K_(d): 2.5 and 3.5 μM, respectively, FIG. 5).

Structural Studies Confirm Binding of Marinopyrrole Compounds to theBH3-Binding Site of Mcl-1.

Structural characterization of 1-20 and II-4 was performed using NMR.HSQC spectra were collected at different ratios of compound:Mcl-1. NMRstudies confirmed binding to Mcl-1 of 1-20 and 11-4, which show fast tointermediate exchange regime in agreement with the low M bindingaffinity as determined by FQ. In FIG. 6, comparison of the Mcl-1 HSQCspectra with and without compound demonstrates significant chemicalshift changes particularly in residues of the BH3-binding site of Mcl-1structure. The residues that undergo chemical shift changes uponcompounds' binding onto the structure of Mcl-1 and the compounds'binding location were mapped (FIG. 6). An induced fit molecular dockingapproach guided by the NMR chemical shifts was used to determine thestructural models of the compounds bound to Mcl-1 using Glide software(Schrodinger; FIG. 6). Structural details of the lowest energystructures suggest that compounds II-4 and 1-20 complement theBH3-binding site using an extended conformation and making severalhydrophobic contacts with the hydrophobic groove of Mcl-1 site.Moreover, the marinopyrrole C═O group consistently interacts throughhydrogen bonding with Arg263 and Thr266; two critical residues for thebinding of the BH3 domain to Mcl-1.

Further Studies Assessing 1-2.

The above ELISA, GST-pull down, FQ and NMR in vitro studies demonstratedphysical binding and rich SAR of the marinopyrrole derivatives.Subsequent intact cell studies demonstrated that some analogues such asI-2, I-3, I-5, IV-5, IV-6, IV-7, IV-8, V-4, VI-11 and VI-12 inducedapoptosis in cultured human cancer cells. Among the most potent of thesewas I-2. I-2 is more potent than 1 at inhibiting the binding of Mcl-1and Bcl-xL to Bim not only in vitro as demonstrated by ELISA (FIG. 7A)but also in MDA-MB-468 cancer cells by co-immunoprecipitation anddensitometry quantitation of Bim/Mcl-1 and Bim/Bcl-xL levels (FIG. 7B).Previously, it has been shown that 1 decreases Mcl-1 levels, and here itwas shown that I-2 was also more potent than 1 at decreasing the levelsof Mcl-1 and Bcl-xL and at increasing the levels of the pro-apoptoticproteins Bak and Bad (FIG. 8). I-2 slightly decreased Bim but had littleeffects on Bcl-2 and Bax (FIG. 8). Consistent with these effects on theBcl-2 family of proteins, I-2 was also more potent than 1 at inducingapoptosis (Caspase 3 and PARP cleavage) (FIG. 8) and at inhibitinganchorage-dependent (MTT assay) and -independent (soft agar assay)proliferation in both MDA-MB-468 and A-549 human lung cancer cells(FIGS. 9A and 9B).

Next, it was determined if I-2 is effective at inhibiting the growth ofA-549 xenografts in nude mice (A-549 cells grew much better in nude micethan MDA-MB-468 cells. FIG. 10A shows that the tumor volumes from day 1to day 14 in the vehicle-treated mice increased by an average of 160±10%whereas those from I-2-treated mice increased by only 45±30%. Treatmentwith vehicle and I-2 had little effect on the body weights of the mice(FIG. 10B).

Previously, it has been shown that the K562 leukemia cells thatover-express Mcl-1 are more sensitive than the parental cells as well asto those that overexpress Bcl-xL and Bcl-2. However, more recently, ithas also been shown that marinopyrrole A is not Mcl-1 selective. Herein,it was shown that parental MDA-MB-468 cells and the correspondingisogenic cell lines that over express Bim along with either Mcl-1,Bcl-xL or Bcl-2 are equally sensitive to I-2 (IC₅₀ values 0.3 to 0.4 μM)or 1 (IC₅₀ values 1.7 to 2.4 μM). The reason for this inconsistency isnot known, but could be due to the different cell lines used.

In addition, a liquid chromatography mass spectrometry (LC-MS) methodwas developed. The optimized method consists of mixing of 1 and I-2 withmouse blood, extracting the drugs with methanol, and injecting theextracted 1 and I-2 into LC-MS using Agilent 1220 Infinity LC/6120Quadrupole LC/MS with Agilent Zorbax, SBC18 column. FIG. 11 shows theresulting standard curves for 1 and I-2.

Furthermore, incubation of 1 and I-2 in 1 M NaOH for 1, 2, 4, 8, 24 and48 hours at room temperature followed by HPLC analysis as describedabove demonstrated that 1 and I-2 were highly stable. Indeed, up to 8hour incubation resulted in less than 2% loss, and even after 48 hoursof exposure to 1 M NaOH, 76% and 81% of 1 and I-2, respectively,remained intact (FIG. 12).

The compounds and methods of the appended claims are not limited inscope by the specific compounds and methods described herein, which areintended as illustrations of a few aspects of the claims and anycompounds and methods that are functionally equivalent are within thescope of this disclosure. Various modifications of the compounds andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compounds, methods, and aspects of thesecompounds and methods are specifically described, other compounds andmethods and combinations of various features of the compounds andmethods are intended to fall within the scope of the appended claims,even if not specifically recited. Thus a combination of steps, elements,components, or constituents can be explicitly mentioned herein; however,all other combinations of steps, elements, components, and constituentsare included, even though not explicitly stated.

What is claimed is:
 1. A compound of the following structure:

wherein each R⁶, R⁷, R¹³, and R¹⁴ are independently chosen fromhydrogen, halogen, and hydroxyl; R⁵ and R¹² are independently chosenfrom hydrogen, halogen, and substituted or unsubstituted alkyl; R², R⁹,R¹⁸ and R¹⁹ are independently chosen from hydrogen, halogen, hydroxyl,cyano, nitro, substituted or unsubstituted sulfonate, substituted orunsubstituted phosphonate, substituted or unsubstituted amino,substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted cycloalkenyl, substituted or unsubstitutedheteroalkenyl, substituted or unsubstituted heterocycloalkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedheteroalkynyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted alkoxyl,substituted or unsubstituted aryloxyl, and substituted or unsubstitutedcarboxyl; or pharmaceutically acceptable salts thereof.
 2. The compoundof claim 1, wherein R⁶, R⁷, R¹³ and R¹⁴ are each independently chosenfrom F, Cl, Br, and I.
 3. The compound of claim 1, wherein R⁶, R⁷, R¹³and R¹⁴ are each Cl.
 4. The compound of claim 1, wherein R⁵ and R¹² areeach H.
 5. The compound of claim 1, having Formula B:

wherein R^(a) and R^(b) are independently chosen from hydrogen, halogen,hydroxyl, cyano, nitro, substituted or unsubstituted sulfonate,substituted or unsubstituted phosphonate, substituted or unsubstitutedamino, substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted cycloalkenyl, substituted or unsubstitutedheteroalkenyl, substituted or unsubstituted heterocycloalkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedheteroalkynyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted alkoxyl,substituted or unsubstituted aryloxyl, and substituted or unsubstitutedcarboxyl; or pharmaceutically acceptable salts thereof.
 6. The compoundof claim 5, wherein R^(a) is chosen from hydrogen and halogen.
 7. Thecompound of claim 5, R^(a) is chosen from hydrogen and Cl.
 8. Thecompound of claim 5, wherein R^(b) is chosen from substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedcarboxyl.
 9. The compound of claim 5, wherein R^(b) is chosen fromCH₂CO₂CH₂CH₃, CH₂CO₂Bu, CH₂CO₂H, CH₂Ph, Ph, cyclohexane, and n-octane.10. The compound of claim 5, Formula B-1:

wherein R^(b) is selected from substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, and substituted or unsubstituted carboxyl; or pharmaceuticallyacceptable salts thereof.
 11. The compound of claim 10, wherein R^(b) ischosen from CH₂CO₂CH₂CH₃, CH₂CO₂Bu, CH₂CO₂H, CH₂Ph, Ph, cyclohexane, andn-octane.
 12. The compound of claim 5, having Formula B-2:

wherein R^(b) is chosen from substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, and substituted or unsubstituted carboxyl; or pharmaceuticallyacceptable salts thereof.
 13. The compound of claim 12, wherein R^(b) ischosen from CH₂CO₂CH₂CH₃, CH₂CO₂Bu, CH₂CO₂H, CH₂Ph, Ph, cyclohexane, andn-octane.
 14. The compound of claim 5, the compound being selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof.
 15. The compound of claim14, wherein the compound is a compound having Formula B-5 or Formula B-7and wherein the butyl group comprises a tert-butyl group.
 16. A methodof treating cancer in a subject, comprising administering to the subjectan effective amount of a compound of claim
 1. 17. The method of claim16, wherein the cancer is selected from the group consisting of bladdercancer, brain cancer, breast cancer, colorectal cancer, cervical cancer,gastrointestinal cancer, genitourinary cancer, head and neck cancer,lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renalcancer, skin cancer, and testicular cancer.
 18. A method of killing atumor cell in a subject, comprising: contacting the tumor cell with aneffective amount of a compound of claim
 1. 19. The method of claim 18,wherein the tumor cell is a Mcl-1 dependent cell.
 20. The method ofclaim 18, further comprising irradiating the tumor cell with aneffective amount of ionizing radiation.